Cell

ABSTRACT

The present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising: (i) an antigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and (iv) an endodomain wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR and wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain.

FIELD OF THE INVENTION

The present invention relates to a cell which comprises more than onechimeric antigen receptor (CAR). The cell may be capable of specificallyrecognising a target cell, due to a differential pattern of expression(or non-expression) of two or more antigens by the target cell.

BACKGROUND TO THE INVENTION

A number of immunotherapeutic agents have been described for use incancer treatment, including therapeutic monoclonal antibodies (mAbs),immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cellengagers.

Typically these immunotherapeutic agents target a single antigen: forinstance, Rituximab targets CD20; Myelotarg targets CD33; andAlemtuzumab targets CD52.

However, it is relatively rare for the presence (or absence) of a singleantigen effectively to describe a cancer, which can lead to a lack ofspecificity.

Most cancers cannot be differentiated from normal tissues on the basisof a single antigen. Hence, considerable “on-target off-tumour” toxicityoccurs whereby normal tissues are damaged by the therapy. For instance,whilst targeting CD20 to treat B-cell lymphomas with Rituximab, theentire normal B-cell compartment is depleted, whilst targeting CD52 totreat chronic lymphocytic leukaemia, the entire lymphoid compartment isdepleted, whilst targeting CD33 to treat acute myeloid leukaemia, theentire myeloid compartment is damaged etc. The predicted problem of“on-target off-tumour” toxicity has been borne out by clinical trials.For example, an approach targeting ERBB2 caused death to a patient withcolon cancer metastatic to the lungs and liver. ERBB2 is over-expressedin colon cancer in some patients, but it is also expressed on severalnormal tissues, including heart and normal vasculature.

For some cancers, targeting the presence of two cancer antigens may bemore selective and therefore effective than targeting one. For example,B-chronic lymphocytic leukaemia (B-CLL) is a common leukaemia which iscurrently treated by targeting CD19. This treats the lymphoma but alsodepletes the entire B-cell compartment such that the treatment has aconsiderable toxic effect. B-CLL has an unusual phenotype in that CD5and CD19 are co-expressed. By targeting only cells which express CD5 andCD19, it would be possible to considerably reduce on-target off-tumourtoxicity.

There is thus a need for immunotherapeutic agents which are capable ofmore targeting to reflect the complex pattern of marker expression thatis associated with many cancers.

Chimeric Antigen Receptors (CARs)

Chimeric antigen receptors are proteins which graft the specificity of amonoclonal antibody (mAb) to the effector function of a T-cell. Theirusual form is that of a type I transmembrane domain protein with anantigen recognizing amino terminus, a spacer, a transmembrane domain allconnected to a compound endodomain which transmits T-cell survival andactivation signals (see FIG. 1A).

The most common form of these molecules are fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies whichrecognize a target antigen, fused via a spacer and a trans-membranedomain to a signaling endodomain. Such molecules result in activation ofthe T-cell in response to recognition by the scFv of its target. When Tcells express such a CAR, they recognize and kill target cells thatexpress the target antigen. Several CARs have been developed againsttumour associated antigens, and adoptive transfer approaches using suchCAR-expressing T cells are currently in clinical trial for the treatmentof various cancers.

However, the use of CAR-expressing T cells is also associated withon-target, off tumour toxicity. For example, a CAR-based approachtargeting carboxy anyhydrase-IX (CAIX) to treat renal cell carcinomaresulted in liver toxicity which is thought to be caused by the specificattack on bile duct epithelial cells (Lamers et al (2013) Mol. Ther.21:904-912).

Dual Targeting CAR Approaches

In order to address the problem of “on target, off tumour” toxicity, CART cells have been developed with dual antigen specificity. In the “dualtargeting” approach, two complementary CARs are co-expressed in the sameT-cell population, each directed to a distant tumour target andengineered to provide complementary signals.

Wlikie et al (2012 J Clin Immunol 32:1059-1070) describe a dualtargeting approach in which ErbB2- and MUC1-specific CARs areco-expressed. The ErbB2-specific CAR provided the CD3ζ signal only andthe MUC1-specific CAR provided the CD28 co-stimulatory signal only. Itwas found that complementary signalling occurred in the presence of bothantigens, leading to IL-2 production. However, IL-2 production wasmodest when compared to control CAR-engineered T cells in whichsignaling is delivered by a fused CD28+CD3i; endodomain.

A similar approach was described by Kloss et at (2013 Nature Biotechnol.31:71-75) in which a CD-19 specific CAR was used which provides aCD3ζ-mediated activation signal in combination with a chimericco-stimulatory receptor specific for PSMA. With this ‘co-CAR’ design,the CAR T-cell receives an activation signal when it encounters a targetcell with one antigen, and a co-stimulatory signal when it encounters atarget cell with the other antigen, and only receives both activatoryand co-stimulatory signals upon encountering target cells bearing bothantigens.

This represents an early attempt at restricting CAR activity to only atarget cell bearing two antigens. This approach however is limited:although CAR T-cell activity will be greatest against targets expressingboth antigens, CAR T-cells will still kill targets expressing onlyantigen recognized by the activatory CAR; further, co-stimulationresults in prolonged effects on T-cells which last long after release oftarget cell. Hence, activity against single-antigen positive T-cellsequal to that against double-positives might be possible for example ina situation where single-positive tissues are adjacent to, or in amigratory path from double positive tumour.

There is thus a need for improved CAR-based therapeutic approaches withreduced on-target off-tumour toxicity where T-cell activation is whollyrestricted to target cells which express both antigens.

DESCRIPTION OF THE FIGURES

FIG. 1: (a) Generalized architecture of a CAR: A binding domainrecognizes antigen; the spacer elevates the binding domain from the cellsurface; the trans-membrane domain anchors the protein to the membraneand the endodomain transmits signals. (b) to (d): Different generationsand permutations of CAR endodomains: (b) initial designs transmittedITAM signals alone through FcεR1-γ or CD3ζ endodomain, while laterdesigns transmitted additional (c) one or (d) two co-stimulatory signalsin cis.

FIG. 2: Schematic diagram illustrating the invention

The invention relates to engineering T-cells to respond to logical rulesof target cell antigen expression. This is best illustrated with animaginary FACS scatter-plot. Target cell populations express both,either or neither of antigens “A” and “B”. Different target populations(marked in red) are killed by T-cells transduced with a pair of CARsconnected by different gates. With OR gated receptors, bothsingle-positive and double-positive cells will be killed. With AND gatedreceptors, only double-positive target cells are killed. With AND NOTgating, double-positive targets are preserved while single-positivetargets

FIG. 3: Creation of target cell populations

SupT1 cells were used as target cells. These cells were transduced toexpress either CD19, CD33 or both CD19 and CD33. Target cells werestained with appropriate antibodies and analysed by flow cytometry.

FIG. 4: Cassette design for an OR gate

A single open reading frame provides both CARs with an in-frame FMD-2Asequence resulting in two proteins. Signal1 is a signal peptide derivedfrom IgG1 (but can be any effective signal peptide). scFv1 is thesingle-chain variable segment which recognizes CD19 (but can be a scFvor peptide loop or ligand or in fact any domain which recognizes anydesired arbitrary target). STK is the CD8 stalk but may be any suitableextracellular domain. CD28tm is the CD28 trans-membrane domain but canby any stable type I protein transmembrane domain and CD3Z is the CD3Zeta endodomain but can be any endodomain which contains ITAMs. Signal2is a signal peptide derived from CD8 but can be any effective signalpeptide which is different in DNA sequence from signal1. scFv recognizesCD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of humanIgG1 but can be any extracellular domain which does not cross-pair withthe spacer used in the first CAR. CD28tm′ and CD3Z′ code for the sameprotein sequence as CD28tm and CD3Z but are codon-wobbled to preventhomologous recombination.

FIG. 5: Schematic representation of the chimeric antigen receptors(CARs) for an OR gate Stimulatory CARs were constructed consisting ofeither an N-terminal A) anti-CD19 scFv domain followed by theextracellular hinge region of human CD8 or B) anti-CD33 scFv domainfollowed by the extracellular hinge, CH2 and CH3 (containing a pvaamutation to reduce FcR binding) region of human IgG1. Both receptorscontain a human CD28 transmembrane domain and a human CD3 Zeta (CD247)intracellular domain. “S” depicts the presence of disulphide bonds.

FIG. 6: Expression data showing co-expression of both CARs on thesurface of one T-cell.

FIG. 7: Functional analysis of the OR gate

Effector cells (5×10̂4 cells) expressing the OR gate construct wereco-incubated with a varying number of target cells and IL-2 was analysedafter 16 hours by ELISA. The graph displays the average maximum IL-2secretion from a chemical stimulation (PMA and lonomycin) of theeffector cells alone and the average background IL-2 from effector cellswithout any stimulus from three replicates.

FIG. 8: Cartoon showing both versions of the cassette used to expressboth AND gates Activating and inhibiting CARs were co-expressed onceagain using a FMD-2A sequence. Signal1 is a signal peptide derived fromIgG1 (but can be any effective signal peptide). scFv1 is thesingle-chain variable segment which recognizes CD19 (but can be a scFvor peptide loop or ligand or in fact any domain which recognizes anydesired arbitrary target). STK is the CD8 stalk but may be any non-bulkyextracellular domain. CD28tm is the CD28 trans-membrane domain but canby any stable type I protein transmembrane domain and CD3Z is the CD3Zeta endodomain but can be any endodomain which contains ITAMs. Signal2is a signal peptide derived from CD8 but can be any effective signalpeptide which is different in DNA sequence from signal1. scFv recognizesCD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of humanIgG1 but can be any bulky extracellular domain. CD45 and CD148 are thetransmembrane and endodomains of CD45 and CD148 respectively but can bederived from any of this class of protein.

FIG. 9: Schematic representation of the protein structure of chimericantigen receptors (CARs) for the AND gates

The stimulatory CAR consisting of an N-terminal anti-CD19 scFv domainfollowed by the extracellular stalk region of human CD8, human CD28transmembrane domain and human CD3 Zeta (CD247) intracellular domain.Two inhibitory CARs were tested. These consist of an N-terminalanti-CD33 scFv domain followed by the extracellular hinge, CH2 and CH3(containing a pvaa mutation to reduce FcR binding) region of human IgG1followed by the transmembrane and intracellular domain of either humanCD148 or CD45. “S” depicts the presence of disulphide bonds.

FIG. 10: Co-expression of activation and inhibitory CARs

BW5147 cells were used as effector cells and were transduced to expressboth the activation anti-CD19 CAR and one of the inhibitory anti-CD33CARs. Effector cells were stained with CD19-mouse-Fc and CD33-rabbit-Fcand with appropriate secondary antibodies and analysed by flowcytometry.

FIG. 11: Functional analysis of the AND gates

Effector cells (5×10̂4 cells) expressing activation anti-CD19 CAR and theinhibitory anti-CD33 CAR with the A) CD148 or B) CXD45 intracellulardomain were co-incubated with a varying number of target cells and IL-2was analysed after 16 hours by ELISA. The graph displays the maximumIL-2 secretion from a chemical stimulation (PMA and lonomycin) of theeffector cells alone and the background IL-2 from effector cells withoutany stimulus from three replicates.

FIG. 12: Cartoon showing three versions of the cassette used to generatethe AND NOT gate

Activating and inhibiting CARs were co-expressed once again using aFMD-2A sequence. Signal1 is a signal peptide derived from IgG1 (but canbe any effective signal peptide).

scFv1 is the single-chain variable segment which recognizes CD19 (butcan be a scFv or peptide loop or ligand or in fact any domain whichrecognizes any desired arbitrary target). STK is the human CD8 stalk butmay be any non-bulky extracellular domain. CD28tm is the CD28trans-membrane domain but can by any stable type I protein transmembranedomain and CD3Z is the CD3 Zeta endodomain but can be any endodomainwhich contains ITAMs. Signal2 is a signal peptide derived from CD8 butcan be any effective signal peptide which is different in DNA sequencefrom signal1. scFv recognizes CD33 but as for scFv1 is arbitrary. muSTKis the mouse CD8 stalk but can be any spacer which co-localises but doesnot cross-pair with that of the activating CAR. dPTPN6 is thephosphatase domain of PTPN6. LAIR1 is the transmembrane and endodomainof LAIR1. 2Aw is a codon-wobbled version of the FMD-2A sequence.SH2-CD148 is the SH2 domain of PTPN6 fused with the phosphatase domainof CD148.

FIG. 13: Schematic representation of the chimeric antigen receptors(CARs) for the NOT AND gates

A) A stimulatory CAR consisting of an N-terminal anti-CD19 scFv domainfollowed by the stalk region of human CD8, human CD28 transmembranedomain and human CD247 intracellular domain. B) An inhibitory CARconsisting of an N-terminal anti-CD33 scFv domain followed by the stalkregion of mouse CD8, transmembrane region of mouse CD8 and thephosphatase domain of PTPN6. C) an inhibitory CAR consisting of anN-terminal anti-CD33 scFv domain followed by the stalk region of mouseCD8 and the transmembrane and intracellular segments of LAIR1. D) Aninhibitory CAR identical to previous CAR except it is co-expressed witha fusion protein of the PTPN6 SH2 domain and the CD148 phosphatasedomain.

FIG. 14: Functional analysis of the NOT AND gate

Effector cells (5×10̂4 cells) expressing the A) full length SHP-1 or B)truncated form of SHP-1 were co-incubated with a varying number oftarget cells and IL-2 was analysed after 16 hours by ELISA. The graphdisplays the average maximum IL-2 secretion from a chemical stimulation(PMA and lonomycin) of the effector cells alone and the averagebackground IL-2 from effector cells without any stimulus from threereplicates.

FIG. 15: Amino acid sequence of an OR gate

FIG. 16: Amino acid sequence of a CD148 and a CD145 based AND gate

FIG. 17: Amino acid sequence of two AND NOT gates

FIG. 18: Dissection of AND gate function

A. The prototype AND gate is illustrated on the right and its functionin response to CD19, CD33 single and CD19, CD33 double positive targetsis shown on the left. B. The scFvs are swapped so the activatingendodomain is triggered by CD33 and the inhibitory endodomain isactivated by CD19. This AND gate remains functional despite this scFvswap. C. The CD8 mouse stalk replaced Fc in the spacer of the inhibitoryCAR. With this modification, the gate fails to respond to either CD19single positive or CD19, CD33 double positive targets.

FIG. 19: Expression of target antigens on artificial target cells

A. Shows flow cytometry scatter plots CD19 vs CD33 of the original setof artificial target cells derived from SupT1 cells. From left to right:double negative SupT1 cells, SupT1 cells positive for CD19, positive forCD33 and positive for both CD19 and CD33. B. Shows flow cytometryscatter plots CD19 vs GD2 of the artificial target cells generated totest the CD19 AND GD2 gate: From left to right: negative SupT1 cells,SupT1 cells expressing CD19, SupT1 cells transduced with GD2 and GM3synthase vectors which become GD2 positive and SupT1 cells transducedwith CD19 as well as GD2 and GM3 synthase which are positive for bothGD2 and CD19. C. Shows flow cytometry scatter plots of CD19 vs EGFRvIIIof the artificial targets generated to test the CD19 AND EGFRvIII gate.From left to right: negative SupT1 cells, SupT1 cells expressing CD19,SupT1 cells transduced with EGFRvIII and SupT1 cells transduced withboth CD19 and EGFRvIII. D. Shows flow cytometry scatter plots of CD19 vsCD5 of the artificial targets generated to test the CD19 AND CD5 gate.From left to right: negative 293T cells, 293T cells transduced withCD19, 293T cells transduced with CD5, 293T cells transduced with bothCD5 and CD19 vectors.

FIG. 20: Generalizability of the AND gate

A. Cartoon of AND gate modified so the second CAR's specificity ischanged from the original specificity of CD33, to generate 3 new CARs:CD19 AND GD2, CD19 AND EGFRvIII, CD19 AND CD5. B. CD19 AND GD2 AND gate:Left: expression of AND gate is shown recombinant CD19-Fc staining(x-axis) for the CD19 CAR, versus anti-human-Fc staining (Y-axis) forthe GD2 CAR. Right: function in response to single positive and doublepositive targets. C. CD19 AND EGFRvIII AND gate: Left: expression of ANDgate is shown recombinant CD19-Fc staining (x-axis) for the CD19 CAR,versus anti-human-Fc staining (Y-axis) for the EGFRvIII CAR. Right:function in response to single positive and double positive targets. D.CD19 AND CD5 AND gate: Left: expression of AND gate is shown recombinantCD19-Fc staining (x-axis) for the CD19 CAR, versus anti-human-Fcstaining (Y-axis) for the CD5 CAR. Right: function in response to singlepositive and double positive targets.

FIG. 21: Function of the AND NOT gates

Function of the three implementations of an AND NOT gate is shown. Acartoon of the gates tested is shown to the right, and function inresponse to single positive and double positive targets is shown to theleft. A. PTPN6 based AND NOT gate whereby the first CAR recognizes CD19,has a human CD8 stalk spacer and an ITAM containing activatingendodomain; is co-expressed with a second CAR that recognizes CD33, hasa mouse CD8 stalk spacer and has an endodomain comprising of a PTPN6phosphatase domain. B. ITIM based AND NOT gate is identical to the PTPN6gate, except the endodomain is replaced by the endodomain from LAIR1. C.CD148 boosted AND NOT gate is identical to the ITIM based gate except anadditional fusion between the PTPN6 SH2 and the endodomain of CD148 isexpressed. All three gates work as expected with activation in responseto CD19 but not in response to CD19 and CD33 together.

FIG. 22: Dissection of PTPN6 based AND NOT gate function

The original PTPN6 based AND NOT gate is compared with several controlsto demonstrate the model. A cartoon of the gates tested is shown to theright, and function in response to single positive and double positivetargets is shown to the left. A. Original AND NOT gate whereby the firstCAR recognizes CD19, has a human CD8 stalk spacer and an ITAM containingactivating endodomain; is co-expressed with a second CAR recognizesCD33, has a mouse CD8 stalk spacer and has an endodomain comprising of aPTPN6 phosphatase domain. B. AND NOT gate modified so the mouse CD8stalk spacer is replaced with an Fc spacer. C AND NOT gate modified sothat the PTPN6 phosphatase domain is replaced with the endodomain fromCD148. Original AND NOT gate (A.) functions as expected triggering inresponse to CD19, but not in response to both CD19 and CD33. The gate inB. triggers both in response to CD19 along or CD19 and CD33 together.The gate in C. does not trigger in response to one or both targets.

FIG. 23: Dissection of LAIR1 based AND NOT gate

Functional activity against CD19 positive, CD33 positive and CD19, CD33double-positive targets is shown. A. Structure and activity of theoriginal ITIM based AND NOT gate. This gate is composed of two CARs: thefirst recognizes CD19, has a human CD8 stalk spacer and an ITAMcontaining endodomain; the second CAR recognizes CD33, has a mouse CD8stalk spacer and an ITIM containing endodomain. B Structure and activityof the control ITIM based gate where the mouse CD8 stalk spacer has beenreplaced by an Fc domain. This gate is composed of two CARs: the firstrecognizes CD19, has a human CD8 stalk spacer and an ITAM containingendodomain; the second CAR recognizes CD33, has an Fc spacer and an ITIMcontaining endodomain. Both gates respond to CD19 single positivetargets, while only the original gate is inactive in response to CD19and CD33 double positive targets.

FIG. 24: Kinetic segregation model of CAR logic gates

Model of kinetic segregation and behaviour of AND gate, NOT AND gate andcontrols. CARs recognize either CD19 or CD33. The immunological synapsecan be imagined between the blue line, which represents the target cellmembrane and the red line, which represents the T-cell membrane. ‘45’ isthe native CD45 protein present on T-cells. ‘H8’ is a CAR ectodomainwith human CD8 stalk as the spacer. ‘Fc’ is a CAR ectodomain with humanHCH2CH3 as the spacer. ‘M8’ is a CAR ectodomain with murine CD8 stalk asthe spacer. ‘19’ represents CD19 on the target cell surface. ‘33’represents CD33 on the target cell surface. The symbol ‘⊕’ represents anactivating endodomain containing (TAMS. The symbol ‘⊖’ represents aphosphatase with slow kinetics—a ‘ligation on’ endodomain such as onecomprising of the catalytic domain of PTPN6 or an ITIM. The symbol ‘Ø’represents a phosphatase with fast kinetics—a ‘ligation off’ endodomainsuch as the endodomain of CD45 or CD148. This symbol is enlarged in thefigure to emphasize its potent activity.

(a) Shows the postulated behaviour of the functional AND gate whichcomprises of a pair of CARs whereby the first CAR recognizes CD19, has ahuman CD8 stalk spacer and an activating endodomain; and the second CARrecognizes CD33, has an Fc spacer and a CD148 endodomain;

(b) Shows the postulated behaviour of the control AND gate. Here, thefirst CAR recognizes CD19, has a human CD8 stalk spacer and anactivating endodomain; and the second CAR recognizes CD33, but has amouse CD8 stalk spacer and a CD148 endodomain;

(c) Shows the behaviour of a functional AND NOT gate which comprises ofa pair of CARs whereby the first CAR recognizes CD19, has a human CD8stalk spacer and an activating endodomain; and the second CAR recognizesCD33, has a mouse CD8 stalk spacer and a PTPN6 endodomain;

(d) Shows the postulated behaviour of the control AND NOT gate whichcomprises of a pair of CARs whereby the first CAR recognizes CD19, has ahuman CD8 stalk spacer and an activating endodomain; and the second CARrecognizes CD33, but has an Fc spacer and a PTPN6 endodomain;

In the first column, target cells are both CD19 and CD33 negative. Inthe second column, targets are CD19 negative and CD33 positive. In thethird column, target cells are CD19 positive and CD33 negative. In thefourth column, target cells are positive for both CD19 and CD33.

FIG. 25: Design of APRIL-based CARs.

The CAR design was modified so that the scFv was replaced with amodified form of A proliferation-inducing ligand (APRIL), whichinteracts with interacts with BCMA, TACI and proteoglycans, to act as anantigen binding domain: APRIL was truncated so that the proteoglycanbinding amino-terminus is absent. A signal peptide was then attached totruncated APRIL amino-terminus to direct the protein to the cellsurface. Three CARs were generated with this APRIL based binding domain:A. In the first CAR, the human CD8 stalk domain was used as a spacerdomain. B. In the second CAR, the hinge from IgG1 was used as a spacerdomain. C. In the third CAR, the hinge, CH2 and CH3 domains of humanIgG1 modified with the pva/a mutations described by Hombach et al (2010Gene Ther. 17:1206-1213) to reduce Fc Receptor binding was used as aspacer (henceforth referred as Fc-pvaa). In all CARs, these spacers wereconnected to the CD28 transmembrane domain and then to a tripartiteendodomain containing a fusion of the CD28, OX40 and the CD3-Zetaendodomain (Pule et al, Molecular therapy, 2005: Volume 12; Issue 5;Pages 933-41).

FIG. 26: Annotated Amino acid sequence of the above three APRIL-CARS

A: Shows the annotated amino acid sequence of the CD8 stalk APRIL CAR;B: Shows the annotated amino acid sequence of the APRIL IgG1 hinge basedCAR; C: Shows the annotated amino acid sequence of the APRIL Fc-pvaabased CAR.

FIG. 27: Expression and ligand binding of different APRIL based CARs

A. The receptors were co-expressed with a marker gene truncated CD34 ina retroviral gene vector. Expression of the marker gene on transducedcells allows confirmation of transduction. B. T-cells were transducedwith APRIL based CARs with either the CD8 stalk spacer, IgG1 hinge or Fcspacer. To test whether these receptors could be stably expressed on thecell surface, T-cells were then stained withanti-APRIL-biotin/Streptavidin APC and anti-CD34. Flow-cytometricanalysis was performed. APRIL was equally detected on the cell surfacein the three CARs suggesting they are equally stably expressed. C. Next,the capacity of the CARs to recognize TACI and BCMA was determined. Thetransduced T-cells were stained with either recombinant BCMA or TACIfused to mouse IgG2a Fc fusion along with an anti-mouse secondary andanti-CD34. All three receptor formats showed binding to both BCMA andTACI. A surprising finding was that binding to BCMA seemed greater thanto TACI. A further surprising finding was that although all three CARswere equally expressed, the CD8 stalk and IgG1 hinge CARs appearedbetter at recognizing BCMA and TACI than that with the Fc spacer.

FIG. 28: Function of the different CAR constructs.

Functional assays were performed with the three different APRIL basedCARs. Normal donor peripheral blood T-cells either non-transduced (NT),or transduced to express the different CARs. Transduction was performedusing equal titer supernatant. These T-cells were then CD56 depleted toremove non-specific NK activity and used as effectors. SupT1 cellseither non-transduced (NT), or transduced to express BCMA or TACI wereused as targets. Data shown is mean and standard deviation from 5independent experiments. A. Specific killing of BCMA and TACI expressingT-cells was determined using Chromium release. B. Interferon-p releasewas also determined. Targets and effectors were co-cultured at a ratioof 1:1. After 24 hours, Interferon-p in the supernatant was assayed byELISA. C. Proliferation/survival of CAR T-cells were also determined bycounting number of CAR T-cells in the same co-culture incubated for afurther 6 days. All 3 CARs direct responses against BCMA and TACIexpressing targets. The responses to BCMA were greater than for TACT.

FIG. 29: AND gate functionality in primary cells

PBMCs were isolated from blood and stimulated using PHA and IL-2. Twodays later the cells were transduced on retronectin coated plates withretro virus containing the CD19:CD33 AND gate construct. On day 5 theexpression level of the two CARs translated by the AND gate constructwas evaluated via flow cytometry and the cells were depleted of CD56+cells (predominantly NK cells). On day 6 the PBMCs were placed in aco-culture with target cells at a 1:2 effector to target cell ratio. Onday 8 the supernatant was collected and analysed for IFN-gamma secretionvia ELISA.

FIG. 30: A selection/hierarchy of possible spacer domains of increasingsize is shown. The ectodomain of CD3-Zeta is suggested as the shortestpossible spacer, followed by the (b) the IgG1 hinge. (c) murine or humanCD8 stalk and the CD28 ectodomains are considered intermediate in sizeand co-segregate. (d) The hinge, CH2 and CH3 domain of IgG1 is biggerand bulkier, and (e) the hinge, CH2, CH3 and CH4 domain of IgM is biggerstill. Given the properties of the target molecules, and the epitope ofthe binding domains on said target molecules, it is possible to use thishierarchy of spacers to create a CAR signaling system which eitherco-segregates or segregates apart upon synapse formation.

FIG. 31: Design rules for building logic gated CAR T-cells.

OR, AND NOT and AND gated CARs are shown in cartoon format with thetarget cell on top, and the T-cell at the bottom with the synapse in themiddle. Target cells express arbitrary target antigens A, and B.

T-cells express two CARs which comprise of anti-A and anti-B recognitiondomains, spacers and endodomains. An OR gate requires (1) spacers simplywhich allow antigen recognition and CAR activation, and (2) both CARs tohave activatory endodomains; An AND NOT gate requires (1) spacers whichresult in co-segregation of both CARs upon recognition of both antigensand (2) one CAR with an activatory endodomain, and the other whoseendodomain comprises or recruits a weak phosphatase; An AND gaterequires (1) spacers which result in segregation of both CARs intodifferent parts of the immunological synapse upon recognition of bothantigens and (2) one CAR with an activatory endodomain, and the otherwhose endodomain comprises of a potent phosphatase.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a panel of “logic-gated” chimericantigen receptor pairs which, when expressed by a cell, such as a Tcell, are capable of detecting a particular pattern of expression of atleast two target antigens. If the at least two target antigens arearbitrarily denoted as antigen A and antigen B, the three possibleoptions are as follows:

“OR GATE”-T cell triggers when either antigen A or antigen B is presenton the target cell “AND GATE”-T cell triggers only when both antigens Aand B are present on the target cell “AND NOT GATE”-T cell triggers ifantigen A is present alone on the target cell, but not if both antigensA and B are present on the target cell Engineered T cells expressingthese CAR combinations can be tailored to be exquisitely specific forcancer cells, based on their particular expression (or lack ofexpression) of two or more markers.

Thus in a first aspect, the present invention provides a cell whichco-expresses a first chimeric antigen receptor (CAR) and second CAR atthe cell surface, each CAR comprising:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer    -   (iii) a trans-membrane domain; and    -   (iv) an intracellular T cell signaling domain (endodomain)        wherein the antigen binding domains of the first and second CARs        bind to different antigens, and wherein the spacer of the first        CAR is different to the spacer of the second CAR, such that the        first and second CARs do not form heterodimers, and wherein        one of the first or second CARs is an activating CAR comprising        an activating intracellular T cell signaling domain and the        other CAR is an inhibitory CAR comprising a “ligation-off” (as        defined herein) inhibitory intracellular T cell signaling        domain.

The cell may be an immune effector cell, such as a T-cell or naturalkiller (NK) cell. Features mentioned herein in connection with a T cellapply equally to other immune effector cells, such as NK cells.

The spacer of the first CAR may have a different length and/or chargeand/or shape and/or configuration and/or glycosylation to the spacer ofthe second CAR, such that when the first CAR and the second CAR bindtheir respective target antigens, the first CAR and second CAR becomespatially separated on the T cell. Ligation of the first and second CARsto their respective antigens causes them to be compartmentalizedtogether or separately in the immunological synapse resulting in controlof activation. This may be understood when one considers the kineticseparation model of T-cell activation (see below).

The first spacer or the second spacer may comprise a CD8 stalk and theother spacer may comprise the hinge, CH2 and CH3 domain of an IgG1.

In the present invention, which relates to the “AND” gate, one of thefirst or second CARs is an activating CAR comprising an activatingendodomain, and the other CAR is a “ligation-off” inhibitory CARcomprising an inhibitory endodomain. The ligation-off inhibitory CARinhibits T-cell activation by the activating CAR in the absence ofinhibitory CAR ligation, but does not significantly inhibit T-cellactivation by the activating CAR when the inhibitory CAR is ligated.Since the spacer of the first CAR has a different length and/or chargeand/or shape and/or configuration and/or glycosylation from the spacerof the second CAR, when both CARs are ligated they segregate. Thiscauses the inhibitory CAR to be spatially separated from the activatingCAR, so that T cell activation can occur. T cell activation thereforeonly occurs in response to a target cell bearing both cognate antigens.

The inhibitory endodomain may comprise all or part of the endodomainfrom a receptor-like tyrosine phosphatase, such as CD148 or CD45.

The antigen-binding domain of the first CAR may bind CD5 and theantigen-binding domain of the second CAR may bind CD19. This is of usein targeting chronic lymphocytic leukaemia (CLL). This disease can betreated by targeting CD19 alone, but at the cost of depleting the entireB-cell compartment. CLL cells are unusual in that they co-express CD5and CD19. Targeting this pair of antigens with an AND gate will increasespecificity and reduce toxicity.

In a second aspect, the present invention provides a nucleic acidsequence encoding both the first and second chimeric antigen receptors(CARs) as defined in the first aspect of the invention.

The nucleic acid sequence according may have the following structure:AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2

in whichAgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR;coexpr is a nucleic acid sequence allowing co-expression of two CARs(e.g. a cleavage site);AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR;which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

The nucleic acid sequence allowing co-expression of two CARs may encodea self-cleaving peptide or a sequence which allows alternative means ofco-expressing two CARs such as an internal ribosome entry sequence or a2^(nd) promoter or other such means whereby one skilled in the art canexpress two proteins from the same vector.

Alternative codons may be used in regions of sequence encoding the sameor similar amino acid sequences, in order to avoid homologousrecombination.

In a third aspect, the present invention provides a kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR) as defined in the first aspect of the        invention, which nucleic acid sequence has the following        structure:        AgB1-spacer1-TM1-endo1        in which        AgB1 is a nucleic acid sequence encoding the antigen-binding        domain of the first CAR;        spacer 1 is a nucleic acid sequence encoding the spacer of the        first CAR;        TM1 is a nucleic acid sequence encoding the transmembrane domain        of the first CAR;        endo 1 is a nucleic acid sequence encoding the endodomain of the        first CAR; and    -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor (CAR) as defined in the first aspect of the        invention, which nucleic acid sequence has the following        structure:        AgB2-spacer2-TM2-endo2        AgB2 is a nucleic acid sequence encoding the antigen-binding        domain of the second CAR;        spacer 2 is a nucleic acid sequence encoding the spacer of the        second CAR;        TM2 is a nucleic acid sequence encoding the transmembrane domain        of the second CAR;        endo 2 is a nucleic acid sequence encoding the endodomain of the        second CAR.

In a fourth aspect, the present invention provides a kit comprising: afirst vector which comprises the first nucleic acid sequence as definedabove; and a second vector which comprises the first nucleic acidsequence as defined above.

The vectors may be plasmid vectors, retroviral vectors or transposonvectors. The vectors may be lentiviral vectors.

In a fifth aspect, the present invention provides a vector comprising anucleic acid sequence according to the second aspect of the invention.The vector may be a lentiviral vector.

The vector may be a plasmid vector, a retroviral vector or a transposonvector.

In a sixth aspect, the present invention involves co-expressing morethan two CARs in such a fashion that a complex pattern of more than twoantigens can be recognized on the target cell.

In a seventh aspect, the present invention provides a method for makinga T cell according to the first aspect of the invention, which comprisesthe step of introducing one or more nucleic acid sequence (s) encodingthe first and second CARs; or one or more vector(s) as defined aboveinto a T cell.

The T cell may be from a sample isolated from a patient, a related orunrelated haematopoietic transplant donor, a completely unconnecteddonor, from cord blood, differentiated from an embryonic cell line,differentiated from an inducible progenitor cell line, or derived from atransformed T cell line.

In an eighth aspect, the present invention provides a pharmaceuticalcomposition comprising a plurality of T cells according to the firstaspect of the invention.

In a ninth aspect, the present invention provides a method for treatingand/or preventing a disease, which comprises the step of administering apharmaceutical composition according to the eighth aspect of theinvention to a subject.

The method may comprise the following steps:

-   -   (i) isolation of a T cell as listed above.    -   (ii) transduction or transfection of the T cells with one or        more nucleic acid sequence(s) encoding the first and second CAR        or one or more vector(s) comprising such nucleic acid        sequence(s); and    -   (iii) administering the T cells from (ii) to the subject.

The disease may be a cancer.

In a tenth aspect, the present invention provides a pharmaceuticalcomposition according to the eighth aspect of the invention for use intreating and/or preventing a disease.

The disease may be a cancer.

In an eleventh aspect, the present invention provides use of a T cellaccording to the first aspect of the invention in the manufacture of amedicament for treating and/or preventing a disease.

The disease may be a cancer.

The present invention also provides a nucleic acid sequence whichcomprises:

a) a first nucleotide sequence encoding a first chimeric antigenreceptor (CAR);b) a second nucleotide sequence encoding a second CAR;c) a sequence encoding a self-cleaving peptide positioned between thefirst and second nucleotide sequences, such that the two CARs areexpressed as separate entities.

Alternative codons may be used in one or more portion(s) of the firstand second nucleotide sequences in regions which encode the same orsimilar amino acid sequence(s).

The present invention also provides a vector and a cell comprising sucha nucleic acid.

The kinetic-segregation based AND gate of the present invention offers asignificant technical advantage to the previously described “co-CAR”,i.e. the dual targeting approach in which two antigens are recognized bytwo CARs which supply either an activating or a co-stimulating signal tothe T-cell.

With the co-CAR approach, although greatest activity might be expectedagainst target cells bearing both antigens, considerable activityagainst tissues bearing only antigen recognized by the activating CARcan be expected. This activity can be expected to be at least that of afirst-generation CAR. First generation CARs have resulted inconsiderable toxicity: for instance biliary toxicity was observed inclinical testing of a first generation CAR recognizing Carbonicanhydrase IX which was unexpectedly expressed on biliary epithelium(Rotterdam ref). Notably, terminally differentiated effectors do notrequire or respond to co-stimulatory signals, so any terminallydifferentiated CAR T-cells would act maximally despite the absence of aco-stimulatory CAR signal.

Further, co-stimulatory signals lead to long-lasting effects on theT-cell population. These effects long outlast the T-cell/target synapseinteraction. Consequently, CAR T-cells which become fully activatedwithin the tumour and migrate could have maximally potent activityagainst single-antigen bearing normal tissues. This “spill-over” effectmay be most pronounced in tissues within, near or which drain from thetumour. In fact, strategies based on the concept of the activity of afirst generation CAR being enhanced by co-stimulatory signals engagednot CAR activation but through a distinct receptor, have been proposedand tested (Rossig, Blood. 2002 Mar. 15; 99(6):2009-16.).

The co-CAR approach hence can be expected to result at best to areduction but not abolition of toxicity towards single antigenexpressing normal tissue. The present invention uses kinetic segregationat the immunological synapse formed between the T-cell/target cell toregulate T-cell triggering itself. Consequently tight absolute controlof triggering in the absence of the second antigen is achieved. Hencethe totality of T-cell activation is restricted to target cellsexpressing both antigens, the AND gate should function irrespective ofthe effector cell type or differentiation state, and no “spill-over”effect AND gate T-cell activation is possible.

Further Aspects of the Invention

The present invention also relates to the aspects listed in thefollowing numbered paragraphs:

1. A T cell which co-expresses a first chimeric antigen receptor (CAR)and second CAR at the cell surface, each CAR comprising:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer    -   (iii) a trans-membrane domain; and    -   (iv) an endodomain        wherein the antigen binding domains of the first and second CARs        bind to different antigens, wherein the spacer of the first CAR        is different to the spacer of the second CAR and wherein one of        the first or second CARs is an activating CAR comprising an        activating endodomain and the other CAR is either an activating        CAR comprising an activating endodomain or an inhibitory CAR        comprising a ligation-on or ligation-off inhibitory endodomain.

2. A T cell according to paragraph 1, wherein the spacer of the firstCAR has a different length and/or charge and/or size and/orconfiguration and/or glycosylation of the spacer of the second CAR, suchthat when the first CAR and the second CAR bind their respective targetantigens, the first CAR and second CAR become spatially separated on theT cell membrane.

3. A T cell according to paragraph 2, wherein either the first spacer orthe second spacer comprises a CD8 stalk and the other spacer comprisesthe hinge, CH2 and CH3 domain of IgG1.

4. A T cell according to paragraph 1, wherein both the first and secondCARs are activating CARs.

5. A T cell according to paragraph 4, wherein one CAR binds CD19 and theother CAR binds CD20.

6. A T cell according to paragraph 2 or 3, wherein one of the first orsecond CARs is an activating CAR comprising an activating endodomain,and the other CAR is an inhibitory CAR comprising a ligation-offinhibitory endodomain, which inhibitory CAR inhibits T-cell activationby the activating CAR in the absence of inhibitory CAR ligation, butdoes not significantly inhibit T-cell activation by the activating CARwhen the inhibitory CAR is ligated.

7. A T cell according to paragraph 6, wherein the inhibitory endodomaincomprises all or part of the endodomain from CD148 or CD45.

8. A T cell according to paragraph 6 or 7, wherein the antigen-bindingdomain of the first CAR binds CD5 and the antigen-binding domain of thesecond CAR binds CD19.

9. A T cell according to paragraph 1 wherein the first and secondspacers are sufficiently different so as to prevent cross-pairing of thefirst and second CARs but are sufficiently similar to result inco-localisation of the first and second CARs following ligation.

10. A T cell according to paragraph 9, wherein one of the first orsecond CARs in an activating CAR comprising an activating endodomain,and the other CAR is an inhibitory CAR comprising a ligation-oninhibitory endodomain, which inhibitory CAR does not significantlyinhibit T-cell activation by the activating CAR in the absence ofinhibitory CAR ligation, but inhibits T-cell activation by theactivating CAR when the inhibitory CAR is ligated.

11. A T cell according to paragraph 10, wherein the ligation-oninhibitory endodomain comprises at least part of a phosphatase.

12. A T cell according to paragraph 11, wherein the ligation-oninhibitory endodomain comprises all or part of PTPN6.

13. A T cell according to paragraph 10, wherein the ligation-oninhibitory endodomain comprises at least one ITIM domain.

14. A T cell according to paragraph 13, wherein activity of theligation-on inhibitory endodomain is enhanced by co-expression of aPTPN6-CD45 or -CD148 fusion protein.

15. A T cell according to any of paragraphs 10 to 14, wherein the CARcomprising the activating endodomain comprises an antigen-binding domainwhich binds CD33 and the CAR which comprises the ligation-on inhibitoryendodomain comprises an antigen-binding domain which binds CD34.

16. A T cell which comprises more than two CARs as defined in thepreceding paragraphs such that it is specifically stimulated by a cell,such as a T cell, bearing a distinct pattern of more than two antigens.

17. A nucleic acid sequence encoding both the first and second chimericantigen receptors (CARs) as defined in any of paragraphs 1 to 16.

18. A nucleic acid sequence according to paragraph 17, which has thefollowing structure:

AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2in whichAgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR;coexpr is a nucleic acid sequence enabling co-expression of both CARsAgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR;which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

19. A nucleic acid sequence according to paragraph 18, wherein coexprencodes a sequence comprising a self-cleaving peptide.

20. A nucleic acid sequence according to paragraph 18 or 19, whereinalternative codons are used in regions of sequence encoding the same orsimilar amino acid sequences, in order to avoid homologousrecombination.

21. A kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR) as defined in any of paragraphs 1 to 16,        which nucleic acid sequence has the following structure:        AgB1-spacer1-TM1-endo1        in which        AgB1 is a nucleic acid sequence encoding the antigen-binding        domain of the first CAR;        spacer 1 is a nucleic acid sequence encoding the spacer of the        first CAR;        TM1 is a nucleic acid sequence encoding the transmembrane domain        of the first CAR;        endo 1 is a nucleic acid sequence encoding the endodomain of the        first CAR; and    -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor (CAR) as defined in any of paragraphs 1 to 16,        which nucleic acid sequence has the following structure:        AgB2-spacer2-TM2-endo2        AgB2 is a nucleic acid sequence encoding the antigen-binding        domain of the second CAR;        spacer 2 is a nucleic acid sequence encoding the spacer of the        second CAR;        TM2 is a nucleic acid sequence encoding the transmembrane domain        of the second CAR;        endo 2 is a nucleic acid sequence encoding the endodomain of the        second CAR.

22. A kit comprising: a first vector which comprises the first nucleicacid sequence as defined in paragraph 21; and a second vector whichcomprises the first nucleic acid sequence as defined in paragraph 21.

23. A kit according to paragraph 22, wherein the vectors are integratingviral vectors or transposons.

24. A vector comprising a nucleic acid sequence according to any ofparagraphs 17 to 20.

25. A retroviral vector or a lentiviral vector or a transposon accordingto paragraph 24.

26. A method for making a T cell according to any of paragraphs 1 to 16,which comprises the step of introducing: a nucleic acid sequenceaccording to any of paragraphs 17 to 20; a first nucleic acid sequenceand a second nucleic acid sequence as defined in paragraph 21; and/or afirst vector and a second vector as defined in paragraph 22 or a vectoraccording to paragraph 24 or 25, into a T cell.

27. A method according to paragraph 24, wherein the T cell is from asample isolated from a subject.

28. A pharmaceutical composition comprising a plurality of T cellsaccording to any of paragraphs 1 to 16.

29. A method for treating and/or preventing a disease, which comprisesthe step of administering a pharmaceutical composition according toparagraph 28 to a subject.

30. A method according to paragraph 29, which comprises the followingsteps:

-   -   (i) isolation of a T cell-containing sample from a subject;    -   (ii) transduction or transfection of the T cells with: a nucleic        acid sequence according to any of paragraphs 17 to 20; a first        nucleic acid sequence and a second nucleic acid sequence as        defined in paragraph 21; a first vector and a second vector as        defined in paragraph 22 or 23 or a vector according to paragraph        24 or 25; and    -   (iii) administering the T cells from (ii) to a the subject.

31. A method according to paragraph 29 or 30, wherein the disease is acancer.

32. A pharmaceutical composition according to paragraph 28 for use intreating and/or preventing a disease.

33. The use of a T cell according to any of paragraphs 1 to 16 in themanufacture of a medicament for treating and/or preventing a disease.

DETAILED DESCRIPTION Chimeric Antigen Receptors (CARs)

CARs, which are shown schematically in FIG. 1, are chimeric type Itrans-membrane proteins which connect an extracellularantigen-recognizing domain (binder) to an intracellular signallingdomain (endodomain). The binder is typically a single-chain variablefragment (scFv) derived from a monoclonal antibody (mAb), but it can bebased on other formats which comprise an antibody-like antigen bindingsite. A spacer domain is usually necessary to isolate the binder fromthe membrane and to allow it a suitable orientation. A common spacerdomain used is the Fc of IgG1. More compact spacers can suffice e.g. thestalk from CD8a and even just the IgG1 hinge alone, depending on theantigen. A trans-membrane domain anchors the protein in the cellmembrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular partsof either the y chain of the FcεR1 or CD3ζ. Consequently, these firstgeneration receptors transmitted immunological signal 1, which wassufficient to trigger T-cell killing of cognate target cells but failedto fully activate the T-cell to proliferate and survive. To overcomethis limitation, compound endodomains have been constructed: fusion ofthe intracellular part of a T-cell co-stimulatory molecule to that ofCD3ζ results in second generation receptors which can transmit anactivating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used is that ofCD28. This supplies the most potent co-stimulatory signal—namelyimmunological signal 2, which triggers T-cell proliferation. Somereceptors have also been described which include TNF receptor familyendodomains, such as the closely related OX40 and 41BB which transmitsurvival signals. Even more potent third generation CARs have now beendescribed which have endodomains capable of transmitting activation,proliferation and survival signals.

CAR-encoding nucleic acids may be transferred to T cells using, forexample, retroviral vectors. Lentiviral vectors may be employed. In thisway, a large number of cancer-specific T cells can be generated foradoptive cell transfer. When the CAR binds the target-antigen, thisresults in the transmission of an activating signal to the T-cell it isexpressed on. Thus the CAR directs the specificity and cytotoxicity ofthe T cell towards tumour cells expressing the targeted antigen.

The first aspect of the invention relates to a T-cell which co-expressesa first CAR and a second CAR such that a T-cell can recognize a desiredpattern of expression on target cells in the manner of a logic gate asdetailed in the truth tables: table 1, 2 and 3.

Both the first and second (and optionally subsequent) CARs comprise:

(i) an antigen-binding domain;(ii) a spacer;(iii) a transmembrane domain; and(iii) an intracellular domain.

TABLE 1 Truth Table for CAR OR GATE Antigen A Antigen B Response AbsentAbsent No activation Absent Present Activation Present Absent ActivationPresent Present Activation

TABLE 2 Truth Table for CAR AND GATE Antigen A Antigen B Response AbsentAbsent No activation Absent Present No Activation Present Absent NoActivation Present Present Activation

TABLE 3 Truth Table for CAR AND NOT GATE Antigen A Antigen B ResponseAbsent Absent No activation Absent Present No Activation Present AbsentActivation Present Present No Activation

The first and second CAR of the T cell of the present invention may beproduced as a polypeptide comprising both CARs, together with a cleavagesite.

SEQ ID No. 1 to 5 give examples of such polypeptides, which eachcomprise two CARs. The CAR may therefore comprise one or other part ofthe following amino acid sequences, which corresponds to a single CAR.

SEQ ID No 1 is a CAR OR gate which recognizes CD19 OR CD33SEQ ID No 2 Is a CAR AND gate which recognizes CD19 AND CD33 using aCD148 phosphataseSEQ ID No 3 Is an alternative implementation of the CAR AND GATE whichrecognizes CD19 AND CD33 which uses a CD45 phosphataseSEQ ID No 4 Is a CAR AND NOT GATE which recognizes CD19 AND NOT CD33based on PTPN6 phosphataseSEQ ID No 5 Is an alternative implementation of the CAR AND NOT gatewhich recognizes CD19 AND NOT CD33 and is based on an ITIM containingendodomain from LAIR1SEQ ID No 6. Is a further alternative implementation of the CAR AND NOTgate which recognizes CD19 AND NOT CD33 and recruits a PTPN6-CD148fusion protein to an ITIM containing endodomain.

SEQ ID No. 1 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID No. 2MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 3MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKALIAFLAFLIIVTSIALLWLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYWKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEH SVNGPASPALNQGS SEQ IDNo. 4 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYWAPLAGICVALLLSLIITLICYHRSRKRVCKSGGGSFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIY VAIAQFIETTKKKL SEQ IDNo. 5 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILIGVSWFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQ STKPMAESITYAAVARH SEQID No. 6 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARHRAEGRGSLLTCGDVEENPGPWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYSGGGGSFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVT TFGKTNGYIASGS

The CAR may comprise a variant of the CAR-encoding part of the sequenceshown as SEQ ID No. 1, 2, 3, 4, 5 or 6 having at least 80, 85, 90, 95,98 or 99% sequence identity, provided that the variant sequence is a CARhaving the required properties.

Methods of sequence alignment are well known in the art and areaccomplished using suitable alignment programs. The % sequence identityrefers to the percentage of amino acid or nucleotide residues that areidentical in the two sequences when they are optimally aligned.Nucleotide and protein sequence homology or identity may be determinedusing standard algorithms such as a BLAST program (Basic Local AlignmentSearch Tool at the National Center for Biotechnology Information) usingdefault parameters, which is publicly available athttp://blast.ncbi.nlm.nih.gov. Other algorithms for determining sequenceidentity or homology include: LALIGN(http://wvww.ebi.ac.uk/Tools/psa/lalign/ andhttp://www.ebi.ac.uk/Tools/psa/laliqn/nucleotide.html), AMAS (Analysisof Multiply Aligned Sequences, athttp://www.compbio.dundee.ac.uk/Software/Amas/amas.html), FASTA(http://www.ebi.ac.uk/Tools/sss/fasta/), Clustal Omega(http://www.ebi.ac.uk/Tools/msa/clustalo/), SIM(http://web.expasy.org/sim/), and EMBOSS Needle(http://www.ebi.ac.ukfTools/psa/emboss_needle/nucleotide.html).

CAR Logical OR Gate

In this embodiment, the antigen binding domains of the first and secondCARs of the present invention bind to different antigens and both CARscomprise an activating endodomain. Both CARs have different spacerdomains to prevent cross-pairing of the two different receptors. A Tcell can hence be engineered to activate upon recognition of either orboth antigens. This is useful in the field of oncology as indicated bythe Goldie-Coldman hypothesis: sole targeting of a single antigen mayresult in tumour escape by modulation of said antigen due to the highmutation rate inherent in most cancers. By simultaneously targeting twoantigens, the probably of such escape is exponentially reduced.

Various tumour associated antigens are known as shown in the followingTable 4. For a given disease, the first CAR and second CAR may bind totwo different TAAs associated with that disease. In this way, tumourescape by modulating a single antigen is prevented, since a secondantigen is also targeted. For example, when targeting a B-cellmalignancy, both CD19 and CD20 can be simultaneously targeted. In thisembodiment, it is important that the two CARs do not heterodimerize.

TABLE 4 Cancer type TAA Diffuse Large B-cell Lymphoma CD19, CD20 Breastcancer ErbB2, MUC1 AML CD13, CD33 Neuroblastoma GD2, NCAM B-CLL CD19,CD52 Colorectal cancer Folate binding protein, CA-125

Kinetic Segregation Model

Subsequent pairing of CARs to generate the AND gate and the AND NOT gateare based on the kinetic segregation model (KS) of T-cell activation.This is a functional model, backed by experimental data, which explainshow antigen recognition by a T-cell receptor is converted intodown-stream activation signals. Briefly: at the ground state, thesignalling components on the T-cell membrane are in dynamic homeostasiswhereby dephosphorylated ITAMs are favoured over phosphorylated ITAMs.This is due to greater activity of the transmembrane CD45/CD148phosphatases over membrane-tethered kinases such as Ick. When a T-cellengages a target cell through a T-cell receptor (or CAR) recognition ofcognate antigen, tight immunological synapses form. This closejuxtapositioning of the T-cell and target membranes excludes CD45/CD148due to their large ectodomains which cannot fit into the synapse.Segregation of a high concentration of T-cell receptor associated ITAMsand kinases in the synapse, in the absence of phosphatases, leads to astate whereby phosphorylated ITAMs are favoured. ZAP70 recognizes athreshold of phosphorylated ITAMs and propagates a T-cell activationsignal. This advanced understanding of T-cell activation is exploited bythe present invention. In particular, the invention is based on thisunderstanding of how ectodomains of different length and/or bulk and/orcharge and/or configuration and/or glycosylation result in differentialsegregation upon synapse formation.

The CAR Logical AND Gate

In this embodiment, one CAR comprises an activating endodomain and oneCAR comprises an inhibitory endodomain whereby the inhibitory CARconstitutively inhibits the first activating CAR, but upon recognitionof its cognate antigen releases its inhibition of the activating CAR. Inthis manner, a T-cell can be engineered to trigger only if a target cellexpresses both cognate antigens. This behaviour is achieved by theactivating CAR comprising an activating endodomain containing ITAMdomains for example the endodomain of CD3 Zeta, and the inhibitory CARcomprising the endodomain from a phosphatase able to dephosphorylate anITAM (e.g. CD45 or CD148). Crucially, the spacer domains of both CARsare significantly different in size and/or shape and/or charge etc. Whenonly the activating CAR is ligated, the inhibitory CAR is in solution onthe T-cell surface and can diffuse in and out of the synapse inhibitingthe activating CAR. When both CARs are ligated, due to differences inspacer properties, the activating and inhibiting CAR are differentiallysegregated allowing the activating CAR to trigger T-cell activationunhindered by the inhibiting CAR.

This is of considerable utility in the field of cancer therapy.Currently, immunotherapies typically target a single antigen. Mostcancers cannot be differentiated from normal tissues on the basis of asingle antigen. Hence, considerable “on-target off-tumour” toxicityoccurs whereby normal tissues are damaged by the therapy. For instance,whilst targeting CD20 to treat B-cell lymphomas with Rituximab, theentire normal B-cell compartment is depleted. For instance, whilsttargeting CD52 to treat chronic lymphocytic leukaemia, the entirelymphoid compartment is depleted. For instance, whilst targeting CD33 totreat acute myeloid leukaemia, the entire myeloid compartment is damagedetc. By restricting activity to a pair of antigens, much more refinedtargeting, and hence less toxic therapy can be developed. A practicalexample is targeting of CLL which expresses both CD5 and CD19. Only asmall proportion of normal B-cells express both antigens, so theoff-target toxicity of targeting both antigens with a logical AND gateis substantially less than targeting each antigen individually.

The design of the present invention is a considerable improvement onprevious implementation as described by Wilkie et al. ((2012). J. Clin.Immunol. 32, 1059-1070) and then tested in vivo (Kloss et al (2013) Nat.Biotechnol. 31, 71-75). In this implementation, the first CAR comprisesof an activating endodomain, and the second a co-stimulatory domain.This way, a T-cell only receives an activating and co-stimulatory signalwhen both antigens are present. However, the T-cell still will activatein the sole presence of the first antigen resulting in the potential foroff-target toxicity. Further, the implementation of the presentinvention allows for multiple compound linked gates whereby a cell caninterpret a complex pattern of antigens.

TABLE 5 Cancer Type Antigens Chronic Lymphocytic Leukaemia CD5, CD19Neuroblastoma ALK, GD2 Glioma EGFR, Vimentin Multiple myeloma BCMA,CD138 Renal Cell Carcinoma Carbonic anhydrase IX, G250 T-ALL CD2,N-Cadherin Prostate Cancer PSMA, hepsin (or others)

The CAR Logical AND NOT Gate

In this embodiment, one CAR comprises an activating endodomain and oneCAR comprises an inhibitory endodomain such that this inhibitory CAR isonly active when it recognizes its cognate antigen. Hence a T-cellengineered in this manner is activated in response to the sole presenceof the first antigen but is not activated when both antigens arepresent. This invention is implemented by inhibitory CARs with a spacerthat co-localise with the first CAR but either the phosphatase activityof the inhibitory CAR should not be so potent that it inhibits insolution, or the inhibitory endodomain in fact recruits a phosphatasesolely when the inhibitory CAR recognizes its cognate target. Suchendodomains are termed “ligation-on” or semi-inhibitory herein.

This invention is of use in refining targeting when a tumour can bedistinguished from normal tissue by the presence of tumour associatedantigen and the loss of an antigen expressed on normal tissue. The ANDNOT gate is of considerable utility in the field of oncology as itallows targeting of an antigen which is expressed by a normal cell,which normal cell also expresses the antigen recognised by the CARcomprising the activating endodomain. An example of such an antigen isCD33 which is expressed by normal stem cells and acute myeloid leukaemia(AML) cells. CD34 is expressed on stem cells but not typically expressedon AML cells. A T-cell recognizing CD33 AND NOT CD34 would result indestruction of leukaemia cells but sparing of normal stem cells.

Potential antigen pairs for use with AND NOT gates are shown in Table 6.

TABLE 6 Antigen expressed by Normal cell which normal cell but notDisease TAA expresses TAA cancer cell AML CD33 stem cells CD34 MyelomaBCMA Dendritic cells CD1c B-CLL CD160 Natural Killer cells CD56 ProstatePSMA Neural Tissue NCAM cancer Bowel A33 Normal bowel HLA class I cancerepithelium

Compound Gates

The kinetic segregation model with the above components allows compoundgates to be made e.g. a T-cell which triggers in response to patterns ofmore than two target antigens. For example, it is possible to make a Tcell which only triggers when three antigens are present (A AND B ANDC). Here, a cell expresses three CARs, each recognizing antigens A, Band C. One CAR is excitatory and two are inhibitory, which each CARhaving spacer domains which result in differential segregation. Onlywhen all three are ligated, will the T-cell activate. A further example:(A OR B) AND C: here, CARs recognizing antigens A and B are activatingand have spacers which co-localise, while CAR recognizing antigen C isinhibitory and has a spacer which results in different co-segregation. Afurther example (A AND NOT B) AND C: Here CAR against antigen A has anactivating endodomain and co-localises with CAR against antigen B whichhas a conditionally inhibiting endodomain. CAR against antigen C has aspacer who segregates differently from A or B and is inhibitory. Infact, ever more complex boolean logic can be programmed with thesesimple components of the invention with any number of CARs and spacers.

Signal Peptide

The CARs of the T cell of the present invention may comprise a signalpeptide so that when the CAR is expressed inside a cell, such as aT-cell, the nascent protein is directed to the endoplasmic reticulum andsubsequently to the cell surface, where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The signal peptide may comprise the SEQ ID No. 7, 8 or 9 or a variantthereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions,substitutions or additions) provided that the signal peptide stillfunctions to cause cell surface expression of the CAR.

SEQ ID No. 7: MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID No. 7 is compact and highly efficient. Itis predicted to give about 95% cleavage after the terminal glycine,giving efficient removal by signal peptidase.

SEQ ID No. 8: MSLPVTALLLPLALLLHAARP

The signal peptide of SEQ ID No. 8 is derived from IgG1.

SEQ ID No. 9: MAVPTQVLGLLLLWLTDARC

The signal peptide of SEQ ID No. 9 is derived from CD8.

The signal peptide for the first CAR may have a different sequence fromthe signal peptide of the second CAR (and from the 3^(rd) CAR and 4^(th)CAR etc).

Antigen Binding Domain

The antigen binding domain is the portion of the CAR which recognizesantigen. Numerous antigen-binding domains are known in the art,including those based on the antigen binding site of an antibody,antibody mimetics, and T-cell receptors. For example, theantigen-binding domain may comprise: a single-chain variable fragment(scFv) derived from a monoclonal antibody; a natural ligand of thetarget antigen; a peptide with sufficient affinity for the target; asingle domain antibody; an artificial single binder such as a Darpin(designed ankyrin repeat protein); or a single-chain derived from aT-cell receptor.

The antigen binding domain may comprise a domain which is not based onthe antigen binding site of an antibody. For example the antigen bindingdomain may comprise a domain based on a protein/peptide which is asoluble ligand for a tumour cell surface receptor (e.g. a solublepeptide such as a cytokine or a chemokine); or an extracellular domainof a membrane anchored ligand or a receptor for which the binding paircounterpart is expressed on the tumour cell.

Examples 11 to 13 relate to a CAR which binds BCMA, in which the antigenbinding domain comprises APRIL, a ligand for BCMA.

The antigen binding domain may be based on a natural ligand of theantigen.

The antigen binding domain may comprise an affinity peptide from acombinatorial library or a de novo designed affinity protein/peptide.

Spacer Domain

CARs comprise a spacer sequence to connect the antigen-binding domainwith the transmembrane domain and spatially separate the antigen-bindingdomain from the endodomain. A flexible spacer allows the antigen-bindingdomain to orient in different directions to facilitate binding.

In the T cell of the present invention, the first and second CARscomprise different spacer molecules. For example, the spacer sequencemay, for example, comprise an IgG1 Fc region, an IgG1 hinge or a humanCD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprisean alternative linker sequence which has similar length and/or domainspacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. Ahuman IgG1 spacer may be altered to remove Fc binding motifs.

Examples of amino acid sequences for these spacers are given below:

SEQ ID No. 10 (hinge-CH2CH3 of human IgG1)AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID No. 11 (human CD8 stalk):TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI SEQ ID No. 12 (human IgG1hinge): AEPKSPDKTHTCPPCPKDPK SEQ ID No. 13 (CD2 ectodomain)KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD SEQ ID no. 14 (CD34 ectodomain)SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT

Since CARs are typically homodimers (see FIG. 1a ), cross-pairing mayresult in a heterodimeric chimeric antigen receptor. This is undesirablefor various reasons, for example: (1) the epitope may not be at the same“level” on the target cell so that a cross-paired CAR may only be ableto bind to one antigen; (2) the VH and VL from the two different scFvcould swap over and either fail to recognize target or worse recognizean unexpected and unpredicted antigen. For the “OR” gate and the “ANDNOT” gate, the spacer of the first CAR is sufficiently different fromthe spacer of the second CAR in order to avoid cross-pairing. The aminoacid sequence of the first spacer may share less that 50%, 40%, 30% or20% identity at the amino acid level with the second spacer.

In other aspects of the invention (for example the AND gate) it isimportant that the spacer of the first CAR has a different length,and/or charge and/or shape and/or configuration and/or glycosylation,such that when both first and second CARs bind their target antigen, thedifference in spacer charge or dimensions results in spatial separationof the two types of CAR to different parts of the membrane to result inactivation as predicted by the kinetic separation model. In theseaspects, the different length, shape and/or configuration of the spacersis carefully chosen bearing in mind the size and binding epitope on thetarget antigen to allow differential segregation upon cognate targetrecognition. For example the IgG1 Hinge, CD8 stalk, IgG1 Fc, ectodomainof CD34, ectodomain of CD45 are expected to differentially segregate.

Examples of spacer pairs which differentially segregate and aretherefore suitable for use with the AND gate are shown in the followingTable:

Stimulatory CAR spacer Inhibitory CAR spacer Human-CD8STKHuman-IgG-Hinge-CH2CH3 Human-CD3z ectodomain Human-IgG-Hinge-CH2CH3Human-IgG-Hinge Human-IgG-Hinge-CH2CH3 Human-CD28STKHuman-IgG-Hinge-CH2CH3 Human-CD8STK Human-IgM-Hinge-CH2CH3CD4 Human-CD3zectodomain Human-IgM-Hinge-CH2CH3CD4 Human-IgG-HingeHuman-IgM-Hinge-CH2CH3CD4 Human-CD28STK Human-IgM-Hinge-CH2CH3CD4

In other aspects of the invention (for example the AND NOT gate), it isimportant that the spacer be sufficiently different as to preventcross-pairing, but to be sufficiently similar to co-localise. Pairs oforthologous spacer sequences may be employed. Examples are murine andhuman CD8 stalks, or alternatively spacer domains which aremonomeric—for instance the ectodomain of CD2.

Examples of spacer pairs which co-localise and are therefore suitablefor use with the AND NOT gate are shown in the following Table:

Stimulatory CAR spacer Inhibitory CAR spacer Human-CD8aSTK Mouse CD8aSTKHuman-CD28STK Mouse CD8aSTK Human-IgG-Hinge Human-CD3z ectodomainHuman-CD8aSTK Mouse CD28STK Human-CD28STK Mouse CD28STKHuman-IgG-Hinge-CH2CH3 Human-IgM-Hinge-CH2CH3CD4

All the spacer domains mentioned above form homodimers. However themechanism is not limited to using homodimeric receptors and should workwith monomeric receptors as long as the spacer is sufficiently rigid. Anexample of such a spacer is CD2 or truncated CD22.

Transmembrane Domain

The transmembrane domain is the sequence of the CAR that spans themembrane.

A transmembrane domain may be any protein structure which isthermodynamically stable in a membrane. This is typically an alpha helixcomprising of several hydrophobic residues.

The transmembrane domain of any transmembrane protein can be used tosupply the transmembrane portion of the invention. The presence and spanof a transmembrane domain of a protein can be determined by thoseskilled in the art using the TMHMM algorithm(http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that thetransmembrane domain of a protein is a relatively simple structure, i.ea polypeptide sequence predicted to form a hydrophobic alpha helix ofsufficient length to span the membrane, an artificially designed TMdomain may also be used (U.S. Pat. No. 7,052,906 B1 describes synthetictransmembrane components).

The transmembrane domain may be derived from CD28, which gives goodreceptor stability.

Activating Endodomain

The endodomain is the signal-transmission portion of the CAR. Afterantigen recognition, receptors cluster, native CD45 and CD148 areexcluded from the synapse and a signal is transmitted to the cell. Themost commonly used endodomain component is that of CD3-zeta whichcontains 3 ITAMs. This transmits an activation signal to the T cellafter antigen is bound. CD3-zeta may not provide a fully competentactivation signal and additional co-stimulatory signaling may be needed.For example, chimeric CD28 and OX40 can be used with CD3-Zeta totransmit a proliferative/survival signal, or all three can be usedtogether.

Where the T cell of the present invention comprises a CAR with anactivating endodomain, it may comprise the CD3-Zeta endodomain alone,the CD3-Zeta endodomain with that of either CD28 or OX40 or the CD28endodomain and OX40 and CD3-Zeta endodomain.

Any endodomain which contains an ITAM motif can act as an activationendodomain in this invention. Several proteins are known to containendodomains with one or more ITAM motifs. Examples of such proteinsinclude the CD3 epsilon chain, the CD3 gamma chain and the CD3 deltachain to name a few. The ITAM motif can be easily recognized as atyrosine separated from a leucine or isoleucine by any two other aminoacids, giving the signature YxxL/I. Typically, but not always, two ofthese motifs are separated by between 6 and 8 amino acids in the tail ofthe molecule (YxxL/Ix(6-8)YxxL/1). Hence, one skilled in the art canreadily find existing proteins which contain one or more ITAM totransmit an activation signal. Further, given the motif is simple and acomplex secondary structure is not required, one skilled in the art candesign polypeptides containing artificial ITAMs to transmit anactivation signal (see WO 2000063372, which relates to syntheticsignalling molecules).

The transmembrane and intracellular T-cell signalling domain(endodomain) of a CAR with an activating endodomain may comprise thesequence shown as SEQ ID No. 15, 16 or 17 or a variant thereof having atleast 80% sequence identity.

SEQ ID No. 15 Comprising CD28 Transmembrane Domain and CD3 Z Endodomain

FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 16 Comprising CD28 Transmembrane Domain and CD28 and CD3 ZetaEndodomains

FWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 17 Comprising CD28 Transmembrane Domain and CD28, OX40 andCD3 Zeta Endodomains.

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 15, 16 or 17, provided that the sequenceprovides an effective trans-membrane domain and an effectiveintracellular T cell signaling domain.

“Ligation-Off” Inhibitory Endodomain

In the embodiment referred above as the AND gate, one of the CARscomprises an inhibitory endodomain such that the inhibitory CAR inhibitsT-cell activation by the activating CAR in the absence of inhibitory CARligation, but does not significantly inhibit T-cell activation by theactivating CAR when the inhibitory CAR is ligated. This is termed a“ligation-off” inhibitory endodomain.

In this case, the spacer of the inhibitory CAR is of a different length,charge, shape and/or configuration and/or glycosylation from the spacerof the activating CAR, such that when both receptors are ligated, thedifference in spacer dimensions results in isolation of the activatingCARs and the inhibitory CARs in different membrane compartments of theimmunological synapse, so that the activating endodomain is releasedfrom inhibition by the inhibitory endodomain.

The inhibitory endodomains for use in a ligation-off inhibitory CAR maytherefore comprise any sequence which inhibits T-cell signaling by theactivating CAR when it is in the same membrane compartment (i.e. in theabsence of the antigen for the inhibitory CAR) but which does notsignificantly inhibit T cell signaling when it is isolated in a separatepart of the membrane from the inhibitory CAR.

The ligation-off inhibitory endodomain may be or comprise a tyrosinephosphatase, such as a receptor-like tyrosine phosphatase. An inhibitoryendodomain may be or comprise any tyrosine phosphatase that is capableof inhibiting the TCR signalling when only the stimulatory receptor isligated. An inhibitory endodomain may be or comprise any tyrosinephosphatase with a sufficiently fast catalytic rate for phosphorylatedITAMs that is capable of inhibiting the TCR signalling when only thestimulatory receptor is ligated.

For example, the inhibitory endodomain of an AND gate may comprise theendodomain of CD148 or CD45. CD148 and CD45 have been shown to actnaturally on the phosphorylated tyrosines up-stream of TCR signalling.

CD148 is a receptor-like protein tyrosine phosphatase which negativelyregulates TCR signaling by interfering with the phosphorylation andfunction of PLCγ1 and LAT.

CD45 present on all hematopoetic cells, is a protein tyrosinephosphatase which is capable of regulating signal transduction andfunctional responses, again by phosphorylating PLC γ1.

An inhibitory endodomain may comprise all of part of a receptor-liketyrosine phosphatase. The phosphatase may interfere with thephosphorylation and/or function of elements involved in T-cellsignalling, such as PLCγ1 and/or LAT.

The transmembrane and endodomain of CD45 and CD148 is shown as SEQ IDNo. 18 and No. 19 respectively.

SEQ ID 18—CD45 Trans-Membrane and Endodomain Sequence

ALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVWKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS

SEQ ID 19—CD148 Trans-Membrane and Endodomain Sequence

AVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAP VTTFGKTNGYIA

An inhibitory CAR may comprise all or part of SEQ ID No 18 or 19 (forexample, it may comprise the phosphatase function of the endodomain). Itmay comprise a variant of the sequence or part thereof having at least80% sequence identity, as long as the variant retains the capacity tobasally inhibit T cell signalling by the activating CAR.

Other spacers and endodomains may be tested for example using the modelsystem exemplified herein. Target cell populations can be created bytransducing a suitable cell line such as a SupT1 cell line either singlyor doubly to establish cells negative for both antigens (the wild-type),positive for either and positive for both (e.g. CD19−CD33−, CD19+CD33−,CD19−CD33+ and CD19+CD33+). T cells such as the mouse T cell line BW5147which releases IL-2 upon activation may be transduced with pairs of CARsand their ability to function in a logic gate measured throughmeasurement of IL-2 release (for example by ELISA). For example, it isshown in Example 4 that both CD148 and CD45 endodomains can function asinhibitory CARs in combination with an activating CAR containing a CD3Zeta endodomain. These CARs rely upon a short/non-bulky CD8 stalk spaceron one CAR and a bulky Fc spacer on the other CAR to achieve AND gating.When both receptors are ligated, the difference in spacer dimensionsresults in isolation of the different receptors in different membranecompartments, releasing the CD3 Zeta receptor from inhibition by theCD148 or CD45 endodomains. In this way, activation only occurs once bothreceptors are activated. It can be readily seen that this modular systemcan be used to test alternative spacer pairs and inhibitory endodomains.If the spacers do not achieve isolation following ligation of bothreceptors, the inhibition would not be released and so no activationwould occur. If the inhibitory endodomain under test is ineffective,activation would be expected in the presence of ligation of theactivating CAR irrespective of the ligation status of the inhibitoryCAR.

“Ligation-on” Endodomain

In the embodiment referred above as the AND NOT gate, one of the CARscomprises a “ligation-on” inhibitory endodomain such that the inhibitoryCAR does not significantly inhibit T-cell activation by the activatingCAR in the absence of inhibitory CAR ligation, but inhibits T-cellactivation by the activating CAR when the inhibitory CAR is ligated.

The “ligation-on” inhibitory endodomain may be or comprise a tyrosinephosphatase that is incapable of inhibiting the TCR signalling when onlythe stimulatory receptor is ligated.

The “ligation-on” inhibitory endodomain may be or comprise a tyrosinephosphatase with a sufficiently slow catalytic rate for phosphorylatedITAMs that is incapable of inhibiting the TCR signalling when only thestimulatory receptor is ligated but it is capable of inhibiting the TCRsignalling response when concentrated at the synapse. Concentration atthe synapse is achieved through inhibitory receptor ligation.

If a tyrosine phosphatase has a catalytic rate which is too fast for a“ligation-on” inhibitory endodomain, then it is possible to tune-downthe catalytic rates of phosphatase through modification such as pointmutations and short linkers (which cause steric hindrance) to make itsuitable for a “ligation-on” inhibitory endodomain.

In this first embodiment the endodomain may be or comprise a phosphatasewhich is considerably less active than CD45 or CD148, such thatsignificant dephosphorylation of ITAMS only occurs when activating andinhibitory endodomains are co-localised. Many suitable sequences areknown in the art. For example, the inhibitory endodomain of a NOT ANDgate may comprise all or part of a protein-tyrosine phosphatase such asPTPN6.

Protein tyrosine phosphatases (PTPs) are signaling molecules thatregulate a variety of cellular processes including cell growth,differentiation, mitotic cycle, and oncogenic transformation. TheN-terminal part of this PTP contains two tandem Src homolog (SH2)domains, which act as protein phospho-tyrosine binding domains, andmediate the interaction of this PTP with its substrates. This PTP isexpressed primarily in hematopoietic cells, and functions as animportant regulator of multiple signaling pathways in hematopoieticcells.

The inhibitor domain may comprise all of PTPN6 (SEQ ID No. 20) or justthe phosphatase domain (SEQ ID No. 21).

SEQ ID 20—Sequence of PTPN6

MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK

SEQ ID 21—Sequence of Phosphatase Domain of PTPN6

FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQF

A second embodiment of a ligation-on inhibitory endodomain is an ITIM(Immunoreceptor Tyrosine-based Inhibition motif) containing endodomainsuch as that from CD22, LAIR-1, the Killer inhibitory receptor family(KIR), LILRB1, CTLA4, PD-1, BTLA etc. When phosphorylated, ITIMsrecruits endogenous PTPN6 through its SH2 domain. If co-localised withan ITAM containing endodomain, dephosphorylation occurs and theactivating CAR is inhibited.

An ITIM is a conserved sequence of amino acids (S/IN/LxYxxI/V/L) that isfound in the cytoplasmic tails of many inhibitory receptors of theimmune system. One skilled in the art can easily find protein domainscontaining an ITIM. A list of human candidate ITIM-containing proteinshas been generated by proteome-wide scans (Staub, et at (2004) Cell.Signal. 16, 435-456). Further, since the consensus sequence is wellknown and little secondary structure appears to be required, one skilledin the art could generate an artificial ITIM.

ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1,KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3 are shown in SEQ ID 22 to 31respectively

PDCD1 endodomain SEQ ID 22CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL BTLA4 SEQ ID 23KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH LILRB1 SEQ ID 24LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS QEGPSPAVPSIYATLAIHLAIR1 SEQ ID 25 HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVAR H CTLA4 SEQ ID 26FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPEC EKQFQPYFIPIN KIR2DL1SEQ ID 27 GNSRHLHVLIGTSWIIPFAILLFFLLHRWCANKKNAWMDQEPAGNRTVNREDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAE SRSKVVSCP KIR2DL4 SEQID 28 GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRTVNREDSDEQDPQEVTYAQLDHCIFTQRKITGPSQRSKRPSTDTSVCIELPNAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPAAGI KIR2DL5 SEQ ID 29TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI KIR3DL1 SEQ ID 30KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTANSEDSDEQDPEEVTYAQLDHCVFTQRKITRPSQRPKTPPTDTILYTELPN AKPRSKVVSCP KIR3DL3SEQ ID 31 KDPGNSRHLHVLIGTSWIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYAQLNHCVFTQRKITRPSQRPKTPPTDTSV

A third embodiment of a ligation-on inhibitory endodomain is an ITIMcontaining endodomain co-expressed with a fusion protein. The fusionprotein may comprise at least part of a protein-tyrosine phosphatase andat least part of a receptor-like tyrosine phosphatase. The fusion maycomprise one or more SH2 domains from the protein-tyrosine phosphatase.For example, the fusion may be between a PTPN6 SH2 domain and CD45endodomain or between a PTPN6 SH2 domain and CD148 endodomain. Whenphosphorylated, the ITIM domains recruit the fusion protein bring thehighly potent CD45 or CD148 phosphatase to proximity to the activatingendodomain blocking activation.

SEQUENCES of Fusion Proteins are Listed 32 and 33

PTPN6-CD45 fusion protein SEQ ID 32WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFMIQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPAS PALNQGS PTPN6-CD148fusion SEQ ID 33 ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA

A ligation-on inhibitory CAR may comprise all or part of SEQ ID No 20 or21. It may comprise all or part of SEQ ID 22 to 31. It may comprise allor part of SEQ ID 22 to 31 co-expressed with either SEQ ID 32 or 33. Itmay comprise a variant of the sequence or part thereof having at least80% sequence identity, as long as the variant retains the capacity toinhibit T cell signaling by the activating CAR upon ligation of theinhibitory CAR.

As above, alternative spacers and endodomains may be tested for exampleusing the model system exemplified herein. It is shown in Example 5 thatthe PTPN6 endodomain can function as a semi-inhibitory CAR incombination with an activating CAR containing a CD3 Zeta endodomain.These CARs rely upon a human CD8 stalk spacer on one CAR and a mouse CD8stalk spacer on the other CAR. The orthologous sequences prevent crosspairing. However, when both receptors are ligated, the similaritybetween the spacers results in co-segregation of the different receptorsin the same membrane compartments. This results in inhibition of the CD3Zeta receptor by the PTPN6 endodomain. If only the activating CAR isligated the PTPN6 endodomain is not sufficiently active to prevent Tcell activation. In this way, activation only occurs if the activatingCAR is ligated and the inhibitory CAR is not ligated (AND NOT gating).It can be readily seen that this modular system can be used to testalternative spacer pairs and inhibitory domains. If the spacers do notachieve co-segregation following ligation of both receptors, theinhibition would not be effective and so activation would occur. If thesemi-inhibitory endodomain under test is ineffective, activation wouldbe expected in the presence of ligation of the activating CARirrespective of the ligation status of the semi-inhibitory CAR.

Co-Expression Site

The second aspect of the invention relates to a nucleic acid whichencodes the first and second CARs.

The nucleic acid may produce a polypeptide which comprises the two CARmolecules joined by a cleavage site. The cleavage site may beself-cleaving, such that when the polypeptide is produced, it isimmediately cleaved into the first and second CARs without the need forany external cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouthdisease virus (FMDV) 2a self-cleaving peptide, which has the sequenceshown as SEQ ID No. 34:

SEQ ID No. 34 RAEGRGSLLTCGDVEENPGP.

The co-expressing sequence may be an internal ribosome entry sequence(IRES). The co-expressing sequence may be an internal promoter.

Cell

The first aspect of the invention relates to a cell which co-expresses afirst CAR and a second CAR at the cell surface.

The cell may be any eukaryotic cell capable of expressing a CAR at thecell surface, such as an immunological cell.

In particular the cell may be an immune effector cell such as a T cellor a natural killer (NK) cell

T cells or T lymphocytes are a type of lymphocyte that play a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. Through IL-10, adenosine and othermolecules secreted by regulatory T cells, the CD8+ cells can beinactivated to an anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Tregcells) arise in the thymus and have been linked to interactions betweendeveloping T cells with both myeloid (CD11c+) and plasmacytoid (CD123+)dendritic cells that have been activated with TSLP. Naturally occurringTreg cells can be distinguished from other T cells by the presence of anintracellular molecule called FoxP3. Mutations of the FOXP3 gene canprevent regulatory T cell development, causing the fatal autoimmunedisease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originateduring a normal immune response.

The T cell of the invention may be any of the T cell types mentionedabove, in particular a CTL.

Natural killer (NK) cells are a type of cytolytic cell which forms partof the innate immune system. NK cells provide rapid responses to innatesignals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are definedas large granular lymphocytes (LGL) and constitute the third kind ofcells differentiated from the common lymphoid progenitor generating Band T lymphocytes. NK cells are known to differentiate and mature in thebone marrow, lymph node, spleen, tonsils and thymus where they thenenter into the circulation.

The CAR cells of the invention may be any of the cell types mentionedabove.

CAR-expressing cells, such as CAR-expressing T or NK cells, may eitherbe created ex vivo either from a patient's own peripheral blood (1^(st)party), or in the setting of a haematopoietic stem cell transplant fromdonor peripheral blood (2^(nd) party), or peripheral blood from anunconnected donor (3^(rd) party).

The present invention also provide a cell composition comprising CARexpressing T cells and/or CAR expressing NK cells according to thepresent invention. The cell composition may be made by transducing ortransfecting a blood-sample ex vivo with a nucleic acid according to thepresent invention.

Alternatively, CAR-expressing cells may be derived from ex vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to the relevant cell type, such as T cells. Alternatively, animmortalized cell line such as a T-cell line which retains its lyticfunction and could act as a therapeutic may be used.

In all these embodiments, CAR cells are generated by introducing DNA orRNA coding for the CARs by one of many means including transduction witha viral vector, transfection with DNA or RNA.

A CAR T cell of the invention may be an ex vivo T cell from a subject.The T cell may be from a peripheral blood mononuclear cell (PBMC)sample. T cells may be activated and/or expanded prior to beingtransduced with CAR-encoding nucleic acid, for example by treatment withan anti-CD3 monoclonal antibody.

A CAR T cell of the invention may be made by:

-   -   (i) isolation of a T cell-containing sample from a subject or        other sources listed above; and    -   (ii) transduction or transfection of the T cells with one or        more nucleic acid sequence(s) encoding the first and second CAR.

The T cells may then by purified, for example, selected on the basis ofco-expression of the first and second CAR.

Nucleic Acid Sequences

The second aspect of the invention relates to one or more nucleic acidsequence(s) which codes for a first CAR and a second CAR as defined inthe first aspect of the invention.

The nucleic acid sequence may comprise one of the following sequences,or a variant thereof

SEQ ID 35 OR gateSEQ ID 36 AND gate using CD45SEQ ID 37 AND gate using CD148SEQ ID 38 AND NOT gate using PTPN6 as endodomainSEQ ID 39 AND NOT gate using LAIR1 endodomainSEQ ID 40 AND NOT gate using LAIR1 and PTPN6 SH2 fusion with CD148phosphatase

SEQ ID No. 35: >MP13974.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-CD28tmZwATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCTCTCCTGGTGACCGTGGCCTTCATCATCTTTTGGGTGCGCTCCCGGGTGAAGTTTTCTCGCTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACCTGGGCAGGCGGGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGACAAAATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGGGCCACGACGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTGCACATGCAGGCCCTGCCACCCAGATGA SEQ ID No.36 >MP14802.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-dCD45ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCACTGATAGCATTTCTGGCATTTCTGATTATTGTGACATCAATAGCCCTGCTTGTTGTTCTCTACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTTAGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGATTGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTATAAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATTATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCACAAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGCCACAGTTATTGTCATGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAATACTGGCCGTCAATGGAAGAGGGCACTCGGGCTTTTGGAGATGTTGTTGTAAAGATCAACCAGCACAAAAGATGTCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAAAAAGAAAAAGCAACTGGAAGAGAGGTGACTCACATTCAGTTCACCAGCTGGCCAGACCACGGGGTGCCTGAGGATCCTCACTTGCTCCTCAAACTGAGAAGGAGAGTGAATGCCTTCAGCAATTTCTTCAGTGGTCCCATTGTGGTGCACTGCAGTGCTGGTGTTGGGCGCACAGGAACCTATATCGGAATTGATGCCATGCTAGAAGGCCTGGAAGCCGAGAACAAAGTGGATGTTTATGGTTATGTTGTCAAGCTAAGGCGACAGAGATGCCTGATGGTTCAAGTAGAGGCCCAGTACATCTTGATCCATCAGGCTTTGGTGGAATACAATCAGTTTGGAGAAACAGAAGTGAATTTGTCTGAATTACATCCATATCTACATAACATGAAGAAAAGGGATCCACCCAGTGAGCCGTCTCCACTAGAGGCTGAATTCCAGAGACTTCCTTCATATAGGAGCTGGAGGACACAGCACATTGGAAATCAAGAAGAAAATAAAAGTAAAAACAGGAATTCTAATGTCATCCCATATGACTATAACAGAGTGCCACTTAAACATGAGCTGGAAATGAGTAAAGAGAGTGAGCATGATTCAGATGAATCCTCTGATGATGACAGTGATTCAGAGGAACCAAGCAAATACATCAATGCATCTTTTATAATGAGCTACTGGAAACCTGAAGTGATGATTGCTGCTCAGGGACCACTGAAGGAGACCATTGGTGACTTTTGGCAGATGATCTTCCAAAGAAAAGTCAAAGTTATTGTTATGCTGACAGAACTGAAACATGGAGACCAGGAAATCTGTGCTCAGTACTGGGGAGAAGGAAAGCAAACATATGGAGATATTGAAGTTGACCTGAAAGACACAGACAAATCTTCAACTTATACCCTTCGTGTCTTTGAACTGAGACATTCCAAGAGGAAAGACTCTCGAACTGTGTACCAGTACCAATATACAAACTGGAGTGTGGAGCAGCTTCCTGCAGAACCCAAGGAATTAATCTCTATGATTCAGGTCGTCAAACAAAAACTTCCCCAGAAGAATTCCTCTGAAGGGAACAAGCATCACAAGAGTACACCTCTACTCATTCACTGCAGGGATGGATCTCAGCAAACGGGAATATTTTGTGCTTTGTTAAATCTCTTAGAAAGTGCGGAAACAGAAGAGGTAGTGGATATTTTTCAAGTGGTAAAAGCTCTACGCAAAGCTAGGCCAGGCATGGTTTCCACATTCGAGCAATATCAATTCCTATATGACGTCATTGCCAGCACCTACCCTGCTCAGAATGGACAAGTAAAGAAAAACAACCATCAAGAAGATAAAATTGAATTTGATAATGAAGTGGACAAAGTAAAGCAGGATGCTAATTGTGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAAGGAACAGGCTGAAGGTTCTGAACCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTCAATGGTCCTGCAAGTCCAGCTTTAAATCAAGGTTC ATAG SEQ ID No.37: >MP14801.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCC TAA SEQ ID No.38 >16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A- aCD33glx-muCD8STK-tm-dPTPN6ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATCTACTGGGCACCTCTGGCCGGAATATGCGTGGCACTGCTGCTGAGCCTCATCATCACCCTGATCTGTTATCACCGAAGCCGCAAGCGGGTGTGTAAAAGTGGAGGCGGAAGCTTCTGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCGCTACAAGAACATTCTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGGACGGGACAGTAACATCCCCGGGTCCGACTACATCAATGCCAACTACATCAAGAACCAGCTGCTAGGCCCTGATGAGAACGCTAAGACCTACATCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAATGACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATGACCACCCGAGAGGTGGAGAAAGGCCGGAACAAATGCGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTGCTTATGGGCCCTACTCTGTGACCAACTGCGGGGAGCATGACACAACCGAATACAAACTCCGTACCTTACAGGTCTCCCCGCTGGACAATGGAGACCTGATTCGGGAGATCTGGCATTACCAGTACCTGAGCTGGCCCGACCACGGGGTCCCCAGTGAGCCTGGGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCATCGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCACCATCATTGTCATCGACATGCTCATGGAGAACATCTCCACCAAGGGCCTGGACTGTGACATTGACATCCAGAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGGGCATGGTGCAGACGGAGGCGCAGTACAAGTTCATCTACGTGGCCATCGCCCAGTTCATTGAAACCACTAAGAAGAAGCTGTGA SEQ ID No.39 >MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-endoATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAG ACACTGA SEQ ID no.40 >MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-endo-2A- PTPN6_SH2-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACAGGGCAGAAGGAAGAGGTAGCCTGCTGACTTGCGGGGACGTGGAAGAGAACCCAGGGCCATGGTATCATGGCCACATGTCTGGCGGGCAGGCAGAGACGCTGCTGCAGGCCAAGGGCGAGCCCTGGACGTTTCTTGTGCGTGAGAGCCTCAGCCAGCCTGGAGACTTCGTGCTTTCTGTGCTCAGTGACCAGCCCAAGGCTGGCCCAGGCTCCCCGCTCAGGGTCACCCACATCAAGGTCATGTGCGAGGGTGGACGCTACACAGTGGGTGGTTTGGAGACCTTCGACAGCCTCACGGACCTGGTGGAGCATTTCAAGAAGACGGGGATTGAGGAGGCCTCAGGCGCCTTTGTCTACCTGCGGCAGCCGTACAGCGGTGGCGGTGGCAGCTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCAGCGGTAGCTAA

The nucleic acid sequence may encode the same amino acid sequence asthat encoded by SEQ ID No. 35, 36, 37, 38, 39 or 40, but may have adifferent nucleic acid sequence, due to the degeneracy of the geneticcode. The nucleic acid sequence may have at least 80, 85, 90, 95, 98 or99% identity to the sequence shown as SEQ ID No. 35, 36, 37, 38, 39 or40, provided that it encodes a first CAR and a second CAR as defined inthe first aspect of the invention.

Vector

The present invention also provides a vector, or kit of vectors whichcomprises one or more CAR-encoding nucleic acid sequence(s). Such avector may be used to introduce the nucleic acid sequence(s) into a hostcell so that it expresses the first and second CARs.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a T cell.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a plurality of CAR-expressing cells, such as T cells or NKcells according to the first aspect of the invention. The pharmaceuticalcomposition may additionally comprise a pharmaceutically acceptablecarrier, diluent or excipient. The pharmaceutical composition mayoptionally comprise one or more further pharmaceutically activepolypeptides and/or compounds. Such a formulation may, for example, bein a form suitable for intravenous infusion.

Method of Treatment

The T cells of the present invention may be capable of killing targetcells, such as cancer cells. The target cell may be recognisable by adefined pattern of antigen expression, for example the expression ofantigen A AND antigen B; the expression of antigen A OR antigen B; orthe expression of antigen A AND NOT antigen B or complex iterations ofthese gates.

T cells of the present invention may be used for the treatment of aninfection, such as a viral infection.

T cells of the invention may also be used for the control of pathogenicimmune responses, for example in autoimmune diseases, allergies andgraft-vs-host rejection.

T cells of the invention may be used for the treatment of a cancerousdisease, such as bladder cancer, breast cancer, colon cancer,endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer,melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer andthyroid cancer.

It is particularly suited for treatment of solid tumours where theavailability of good selective single targets is limited.

T cells of the invention may be used to treat: cancers of the oralcavity and pharynx which includes cancer of the tongue, mouth andpharynx; cancers of the digestive system which includes oesophageal,gastric and colorectal cancers; cancers of the liver and biliary treewhich includes hepatocellular carcinomas and cholangiocarcinomas;cancers of the respiratory system which includes bronchogenic cancersand cancers of the larynx; cancers of bone and joints which includesosteosarcoma; cancers of the skin which includes melanoma; breastcancer; cancers of the genital tract which include uterine, ovarian andcervical cancer in women, prostate and testicular cancer in men; cancersof the renal tract which include renal cell carcinoma and transitionalcell carcinomas of the utterers or bladder; brain cancers includinggliomas, glioblastoma multiforme and medullobastomas; cancers of theendocrine system including thyroid cancer, adrenal carcinoma and cancersassociated with multiple endocrine neoplasm syndromes; lymphomasincluding Hodgkin's lymphoma and non-Hodgkin lymphoma; Multiple Myelomaand plasmacytomas; leukaemias both acute and chronic, myeloid orlymphoid; and cancers of other and unspecified sites includingneuroblastoma.

Treatment with the T cells of the invention may help prevent the escapeor release of tumour cells which often occurs with standard approaches.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 Creation of Target Cell Populations

For the purposes of proving the principle of the invention, receptorsbased on anti-CD19 and anti-CD33 were arbitrarily chosen. Usingretroviral vectors, CD19 and CD33 were cloned. These proteins weretruncated so that they do not signal and could be stably expressed forprolonged periods. Next, these vectors were used to transduce the SupT1cell line either singly or doubly to establish cells negative for bothantigen (the wild-type), positive for either and positive for both. Theexpression data are shown in FIG. 3.

Example 2 Design and Function of the OR Gate

To construct the OR gate, a pair of receptors recognizing CD19 and CD33were co-expressed. Different spacers were used to prevent cross-pairing.Both receptors had a trans-membrane domain derived from CD28 to improvesurface stability and an endodomain derived from that of CD3 Zeta toprovide a simple activating signal. In this way, a pair of independent1^(st) generation CARs were co-expressed. The retroviral vector cassetteused to co-express the sequences utilizes a foot-and-mouth 2Aself-cleaving peptide to allow co-expression 1:1 of both receptors. Thecassette design is shown in FIG. 4, and the protein structures in FIG.5. The nucleotide sequence of homologous regions was codon-wobbled toprevent recombination during retroviral vector reverse transcription.

Example 3 Testing the OR Gate

Expression of both CARs was tested on the T-cell surface by stainingwith cognate antigen fused to Fc. By using different species of Fcdomains (mouse for CD19 and rabbit for CD33), co-expression of both CARswas determined on the cell surface by staining with different secondaryantibodies conjugated with different fluorophores. This is shown in FIG.6.

Functional testing was then carried out using the mouse T-cell lineBW5147. This cell line releases IL2 upon activation allowing a simplequantitative readout. These T-cells were co-cultured with increasingamounts of the artificial target cells described above. T-cellsresponded to target cells expressing either antigen, as shown by IL2release measured by ELISA. Both CARs were shown to be expressed on thecell surfaces and the T-cells were shown to respond to either or bothantigens. These data are show in FIG. 7.

Example 4 Design and Function of the AND Gate

The AND gate combines a simple activating receptor with a receptor whichbasally inhibits activity, but whose inhibition is turned off once thereceptor is ligated. This was achieved by combining a standard 1^(st)generation CAR with a short/non-bulky CD8 stalk spacer and a CD3 Zetaendodomain with a second receptor with a bulky Fc spacer whoseendodomain contained either CD148 or CD45 endodomains. When bothreceptors are ligated, the difference in spacer dimensions results inisolation of the different receptors in different membrane compartments,releasing the CD3 Zeta receptor from inhibition by the CD148 or CD45endodomains. In this way, activation only occurs once both receptors areactivated. CD148 and CD45 were chosen for this as they function in thismanner natively: for instance, the very bulky CD45 ectodomain excludesthe entire receptor from the immunological synapse. The expressioncassette is depicted in FIG. 8 and the subsequent proteins in FIG. 9.

Surface staining for the different specificity showed that both receptorpairs could be effectively expressed on the cell surface shown in FIG.10. Function in BW5147 shows that the T-cell is only activated in thepresence of both antigens (FIG. 11).

Example 5 Demonstration of Generalizability of the AND Gate

To ensure that the observations were not a manifestation of somespecific characteristic of CD19/CD33 and their binders which had beenused, the two targeting scFvs were swapped such that now, the activation(ITAM) signal was transmitted upon recognition of CD33, rather thanCD19; and the inhibitory (CD148) signal was transmitted upon recognitionof CD19, rather than of CD33. Since CD45 and CD148 endodomains areconsidered to be functionally similar, experimentation was restricted toAND gates with CD148 endodomain. This should still result in afunctional AND gate. T-cells expressing the new logic gate wherechallenged with targets bearing either CD19 or CD33 alone, or both. TheT-cells responded to targets expressing both CD19 and CD33, but not totargets expressing only one or none of these antigens. This shows thatthe AND gate is still functional in this format (FIG. 18B).

On the same lines, it was sought to establish how generalizable our ANDgate is: the AND gate should be generalizable across different targets.While there may be lesser or greater fidelity of the gate given relativeantigen density, cognate scFv binding kinetics and precise distance ofthe scFv binding epitope, one would expect to see some AND gatemanifestations with a wide set of targets and binders. To test this,three additional AND gates were generated. Once again, experimentationwas restricted to the CD148 version of the AND gate. The second scFvfrom the original CD148 AND gate was replaced with the anti-GD2 scFvhuK666 (SEQ ID 41 and SEQ ID 42), or with the anti-CD5 scFv (SEQ ID 43and SEQ ID 44), or the anti-EGFRvIII scFv MR1.1 (SEQ ID 45 AND SEQ ID46) to generate the following CAR AND gates: CD19 AND GD2; CD19 AND CD5;CD19 AND EGFRvIII. The following artificial antigen expressing celllines were also generated: by transducing SupT1, and our SupT1.CD19 withGM3 and GD2 synthases SupT1.GD2 and SupT1.CD19.GD2 were generated. Bytransducing SupT1 and SupT1.CD19 with a retroviral vector coding forEGFRvIII SupT1.EGFRvIII and SupT1.CD19.EGFRvIII were generated. SinceCD5 is expressed on SupT1 cells, a different cell line was used togenerate the target cells: 293T cells were generated which express CD19alone, CD5 alone and both CD5 and CD19 together. Expression wasconfirmed by flow-cytometry (FIG. 19). T-cells expressing the three newCAR AND gates were challenged with SupT1.CD19 and respective cognatedouble positive and single positive target cells. All three AND gatesdemonstrated reduced activation by the double positive cell lines incomparison with the single positive targets (FIG. 20). This demonstratesgeneralizability of the AND gate design to arbitrary targets and cognatebinders.

Example 6 Experimental Proof of Kinetic Segregation Model of CAR ANDGate

The aim was to prove the model that differential segregation caused bydifferent spacers is the central mechanism behind the ability togenerate these logic CAR gates. The model is that if only the activatingCAR is ligated, the potent inhibiting ‘ligation off’ type CAR is insolution in the membrane and can inhibit the activating CAR. Once bothCARs are ligated, if both CAR spacers are sufficiently different, theywill segregate within the synapse and not co-localize. Hence, a keyrequirement is that the spacers are sufficiently different. If the modelis correct, if both spacers are sufficiently similar so they co-localizewhen both receptors are ligated, the gate will fail to function. To testthis, the “bulky” Fc spacer in the original CAR we replaced with amurine CD8 spacer. It was predicted that this has the similar length,bulk and charge as human CD8 but so should not cross-pair with it.Hence, the new gate had a first CAR which recognizes CD19, a human CD8stalk spacer and an activatory endodomain; while the second CARrecognizes CD33, has a mouse CD8 stalk spacer and a CD148 endodomain(FIG. 18C). T-cells were transduced to express this new CAR gate. TheseT-cells were then challenged with SupT1 cells expressing CD19 alone,CD33 alone or CD19 and CD33 together. T-cells did not respond to SupT1cells expressing either antigen alone as per the original AND gate.However, CAR T-cells failed to respond to SupT1 cells expressing bothantigens, thereby confirming the model (FIG. 18C). A functional AND gaterequires both CARs to have spacers sufficiently different so that theydo not co-localize within an immunological synapse (FIGS. 23A and B).

Example 7 Design and Function of an AND NOT Gate

Phosphatases such as CD45 and CD148 are so potent that even a smallamount entering an immunological synapse can inhibit ITAM activation.This is the basis of inhibition of the logical AND gate. Other classesof phosphatases are not as potent e.g. PTPN6 and related phosphatases.It was predicted that a small amount of PTPN6 entering a synapse bydiffusion would not inhibit activation. In addition, it was predictedthat if an inhibitory CAR had a sufficiently similar spacer to anactivating CAR, it could co-localize within a synapse if both CARs wereligated. In this case, large amounts of the inhibitory endodomain wouldbe sufficient to stop the ITAMS from activating when both antigens werepresent. In this way, an AND NOT gate could be created.

For the NOT AND gate, the second signal needs to “veto” activation. Thisis done by bringing an inhibitory signal into the immunological synapse,for example by bringing in the phosphatase of an enzyme such as PTPN6.We hence generated an initial AND NOT gate as follows: two CARsco-expressed whereby the first recognizes CD19, has a human CD8 stalkspacer and an activating endodomain; co-expressed with an anti-CD33 CARwith a mouse CD8 stalk spacer and an endodomain comprising of thecatalytic domain of PTPN6 (SEQ ID 38, FIG. 13 A with B). A suitablecassette is shown in FIG. 12 and preliminary functional data are shownin FIG. 14.

In addition, an alternative strategy was developed for generating an ANDNOT gate. Immune Tyrosinase Inhibitory Motifs (ITIMs) are activated in asimilar manner to ITAMS, in that they become phosphorylated by Ick uponclustering and exclusion of phosphatases. Instead of triggeringactivation by binding ZAP70, phosphorylated ITIMs recruit phosphataseslike PTPN6 through their cognate SH2 domains. An ITIM can function as aninhibitory endodomain, as long as the spacers on the activating andinhibiting CARs can co-localize. To generate this construct, an AND NOTgate was generated as follows: two CARs co-expressed—the firstrecognizes CD19, has a human CD8 stalk spacer and an activatingendodomain; co-expressed with an anti-CD33 CAR with a mouse CD8 stalkspacer and an ITIM containing endodomain derived from that of LAIR1 (SEQID 39, FIG. 13 A with C).

A further, more complex AND NOT gate was also developed, whereby an ITIMis enhanced by the presence of an additional chimeric protein: anintracellular fusion of the SH2 domain is of PTPN6 and the endodomain ofCD148. In this design three proteins are expressed—the first recognizesCD19, has a human CD8 stalk spacer and an activating endodomain;co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer and anITIM containing endodomain derived from that of LAIR1. A further 2Apeptide, allows co-expression of the PTPN6-CD148 fusion (SEQ ID 40,FIGS. 13 A and D). It was predicted that these AND NOT gates would havea different range of inhibition: PTPN6-CD148>PTPN6>>ITIM.

T-cells were transduced with these gates and challenged with targetsexpressing either CD19 or CD33 alone, or both CD19 and CD33 together.All three gates responded to targets expressing only CD19, but nottargets expressing both CD19 and CD33 together (FIG. 21), confirmingthat all three of the AND NOT gates were functional.

Example 8 Experimental Proof of Kinetic Segregation Model of PTPN6 BasedAND NOT Gate

The model of the AND NOT gate centres around the fact that the nature ofthe spacers used in both CARs is pivotal for the correct function of thegate. In the functional AND NOT gate with PTPN6, both CAR spacers aresufficiently similar that when both CARs are ligated, both co-localizewithin the synapse so the high concentration even the weak PTPN6 issufficient to inhibit activation. If the spacers were different,segregation in the synapse will isolate the PTPN6 from the ITAM allowingactivation disrupting the AND NOT gate. To test this, a control wasgenerated replacing the murine CD8 stalk spacer with that of Fc. In thiscase, the test gate consisted of two CARs, the first recognizes CD19,has a human CD8 stalk spacer and an ITAM endodomain; while the secondCAR recognizes CD33, has an Fc spacer and an endodomain comprising ofthe phosphatase from PTPN6. This gate activates in response to CD19, butalso activates in response to CD19 and CD33 together (FIG. 22B, wherefunction of this gate is compared with that of the original AND NOT, andthe control AND gate variant described in Example 6). This experimentaldata proves the model that for a functional AND NOT gate with PTPN6,co-localizing spacers are needed.

Example 9 Experimental Proof of Kinetic Segregation Model of ITIM BasedAND NOT Gate

Similar to the PTPN6 based AND NOT gate, the ITIM based gate alsorequires co-localization in an immunological synapse to function as anAND NOT gate. To prove this hypothesis, a control ITIM based gate wasgenerated as follows: two CARs co-expressed—the first recognizes CD19,has a human CD8 stalk spacer and an activating endodomain; co-expressedwith an anti-CD33 CAR with an Fc spacer and an ITIM containingendodomain derived from that of LAIR1. The activity of this gate wascompared with that of the original ITIM based AND NOT gate. In thiscase, the modified gate activated in response to targets expressingCD19, but also activated in response to cells expressing both CD19 andCD33. These data indicate that ITIM based AND NOT gates follow thekinetic segregation based model and a correct spacer must be selected tocreate a functional gate (FIG. 23B).

Example 10 Summary of Model of CAR Logic Gates Generated by KineticSegregation

Based on current understanding of the kinetic-segregation model and theexperimental data described herein, a summary of the model for a two-CARgate is presented in FIG. 24. The Figure shows a cell expressing twoCARs, each recognizing a different antigen. When either or both CARsrecognize a target antigen on a cell, a synapse forms and native CD45and CD148 are excluded from the synapse due to the bulk of theirectodomain. This sets the stage for T-cell activation. In the case thatthe target cell bears only one cognate antigen, the cognate CAR isligated and the cognate CAR segregates into the synapse. The unligatedCAR remains in solution on the T-cell membrane and can diffuse in andout of the synapse so that an area of high local concentration ofligated CAR with low concentration of unligated CAR forms. In this case,if the ligated CAR has an ITAM and the non-ligated CAR has ‘ligationoff’ type inhibitory endodomain such as that of CD148, the amount ofnon-ligated CAR is sufficient to inhibit activation and the gate is off.In contrast, in this case, if the ligated CAR has an ITAM and thenon-ligated CAR has a ‘ligation on’ type inhibitory endodomain such asPTPN6, the amount of non-ligated CAR is insufficient to inhibit and thegate is on. When challenged by a target cell bearing both cognateantigens, both cognate CARs are ligated and form part of animmunological synapse. Importantly, if the CAR spacers are sufficientlysimilar, the CARs co-localize in the synapse but if the CAR spacers aresufficiently different the CARs segregate within the synapse. In thislatter case, areas of membrane form whereby high concentrations of oneCAR are present but the other CAR is absent. In this case sincesegregation is complete, even if the inhibitory endodomain is a‘ligation off’ type, the gate is on. In the former case, areas ofmembrane form with high concentrations of both CARs mixed together. Inthis case, since both endodomains are concentrated, even if theinhibitory endodomain is ‘ligation on’ type, the gate is off. Byselecting the correct combination of spacer and endodomain logic can beprogrammed into a CAR T-cell.

Based on our work above, we have established a series of design rules toallow generation of logic-gated CARs (illustrated in FIG. 31). Togenerate an “antigen A OR antigen B” gated CAR T-cell, anti-A and anti-BCARs must be generated such that (1) each CAR has a spacer which simplyallows antigen access and synapse formation such that the CAR functions,and (2) Each CAR has an activating endodomain; To generate an “antigen AAND NOT B” gated CAR T-cell, anti-A and anti-B CARs must be generatedsuch that (1) both CARs have spacers which do not cross-pair, but whichwill allow the CARs to co-segregate upon recognition of both cognateantigens on the target cell, (2) and one CAR has an activatingendodomain, while the other CAR has an endodomain which comprises orrecruits a weak phosphatase (e.g. PTPN6); (3) To generate an “antigen AAND antigen B” gated CAR T-cell, anti-A and anti-B CARs must begenerated such that (1) one CAR has a spacer sufficiently different fromthe other CAR such that both CARs will not co-segregate upon recognitionof both cognate antigens on the target cell, (2) one CAR has anactivating endodomain, while the other car has an endodomain whichcomprises of a potent phosphatase (e.g. that of CD45 or CD148). Thecorrect spacers to achieve the desired effect can be selected from a setof spacers with known size/shape etc as well as taking intoconsideration size/shape etc of the target antigen (for instance seeFIG. 30) and the location of the cognate epitope on the target antigen.

SEQ ID No 41: SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa- dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 42:SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa- dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCAGGTGCAGCTGCAGGAGTCTGGCCCAGGCCTGGTGAAGCCCAGCCAGACCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGGCCAGCTACAACATCCACTGGGTGCGGCAGCCCCCAGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGCTGGCGGCAGCACCAACTACAACAGCGCCCTGATGAGCCGGCTGACCATCAGCAAGGACAACAGCAAGAACCAGGTGTTCCTGAAGATGAGCAGCCTGACAGCCGCCGACACCGCCGTGTACTACTGCGCCAAGCGGAGCGACGACTACAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGAACCAGATGACCCAGAGCCCCAGCAGCTTGAGCGCCAGCGTGGGCGACCGGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGAGCGGCAAGGCCCCAAAGGTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCGGCTACCCCATCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA SEQ ID No. 43:SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa- dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDVYYNPSLKNQLTISKDASRDQVFLKITNLDTADTATYYCVRRRATGTGFDYWGQGTTLTVSSGGGGSGGGGSGGGGSNIVMTQSHKFMSTSVGDRVSIACKASQDVGTAVAWYQQKPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCHQYNSYNTFGSGTRLELKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 44:SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa- dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCAGGTGACCCTGAAGGAGAGCGGTCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATTCGGCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACGTGTACTACAACCCCAGCCTGAAGAACCAGCTGACCATCAGCAAGGACGCCAGCCGGGACCAGGTGTTCCTGAAGATCACCAACCTGGACACCGCCGACACCGCCACCTACTACTGCGTGCGGCGCCGGGCCACCGGCACCGGCTTCGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGGTGGCGGTGGCAGCGGCGGCGGCGGAAGCGGAGGTGGTGGCAGCAACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACCGGGTGAGCATCGCCTGCAAGGCCAGCCAGGACGTGGGCACCGCCGTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCCAAGCTGCTGATCTACTGGACCAGCACCCGGCACACCGGCGTGCCCGACCGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTACTTCTGCCACCAGTACAACAGCTACAACACCTTCGGCAGCGGCACCCGGCTGGAGCTGAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA SEQ ID No. 45:SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVKLQQSGGGLVKPGASLKLSCVTSGFTFRKFGMSWVRQTSDKRLEWVASISTGGYNTYYSDNVKGRFTISRENAKNTLYLQMSSLKSEDTALYYCTRGYSSTSYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPASLSVATGEKVTIRCMTSTDIDDDMNWYQQKPGEPPKFLISEGNTLRPGVPSRFSSSGTGTDFVFTIENTLSEDVGDYYCLQSFNVPLTFGDGTKLEIKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 46:SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCAGGTGAAGCTGCAGCAGAGCGGCGGAGGCCTGGTGAAGCCCGGCGCCAGCCTGAAGCTGAGCTGCGTGACCAGCGGCTTCACCTTCCGGAAGTTCGGCATGAGCTGGGTGCGGCAGACCAGCGACAAGCGGCTGGAGTGGGTGGCCAGCATCAGCACCGGCGGCTACAACACCTACTACAGCGACAACGTGAAGGGCCGGTTCACCATCAGCCGGGAGAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCGCCCTGTACTACTGCACCCGGGGCTACAGCAGCACCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACAGTGAGCAGCGGCGGAGGAGGCAGTGGTGGGGGTGGATCTGGCGGAGGTGGCAGCGACATCGAGCTGACCCAGAGCCCCGCCAGCCTGAGCGTGGCCACCGGCGAGAAGGTGACCATCCGGTGCATGACCAGCACCGACATCGACGACGACATGAACTGGTACCAGCAGAAGCCCGGCGAGCCCCCAAAGTTCCTGATCAGCGAGGGCAACACCCTGCGGCCCGGCGTGCCCAGCCGGTTCAGCAGCAGCGGCACCGGCACCGACTTCGTGTTCACCATCGAGAACACCCTGAGCGAGGACGTGGGCGACTACTACTGCCTGCAGAGCTTCAACGTGCCCCTGACCTTCGGCGACGGCACCAAGCTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA

Example 11 Design and Construction of APRIL Based CARs

APRIL in its natural form is a secreted type II protein. The use ofAPRIL as a BCMA binding domain for a CAR requires conversion of thistype II secreted protein to a type I membrane bound protein and for thisprotein to be stable and to retain binding to BCMA in this form. Togenerate candidate molecules, the extreme amino-terminus of APRIL wasdeleted to remove binding to proteoglycans. Next, a signal peptide wasadded to direct the nascent protein to the endoplasmic reticulum andhence the cell surface. Also, because the nature of spacer used canalter the function of a CAR, three different spacer domains were tested:an APRIL based CAR was generated comprising (i) a human IgG1 spaceraltered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) theIgG1 hinge alone (cartoon in FIG. 25 and amino acid sequences in FIG.26). These CARs were expressed in a bicistronic retroviral vector (FIG.27A) so that a marker protein-truncated CD34 could be co-expressed as aconvenient marker gene.

Example 12 Expression and Function of APRIL Based CARs

The aim of this study was to test whether the APRIL based CARs which hadbeen constructed were expressed on the cell surface and whether APRILhad folded to form the native protein. T-cells were transduced withthese different CAR constructs and stained using a commerciallyavailable anti-APRIL mAb, along with staining for the marker gene andanalysed by flow-cytometry. The results of this experiment are shown inFIG. 27B where APRIL binding is plotting against marker genefluorescence. These data show that in this format, the APRIL based CARsare expressed on the cell surface and APRIL folds sufficiently to berecognized by an anti-APRIL mAb.

Next, it was determined whether APRIL in this format could recognizeBCMA and TACI. Recombinant BCMA and TACI were generated as fusions withmouse IgG2a-Fc. These recombinant proteins were incubated with thetransduced T-cells. After this, the cells were washed and stained withan anti-mouse fluorophore conjugated antibody and an antibody to detectthe marker gene conjugated to a different fluorophore. The cells wereanalysed by flow cytometry and the results are presented in FIG. 27C.The different CARs were able to bind both BCMA and TACI. Surprisingly,the CARs were better able to bind BCMA than TACI. Also, surprisinglyCARs with a CD8 stalk or IgG1 hinge spacer were better able to bind BCMAand TACI than CAR with an Fc spacer.

Example 13 APRIL Based Chimeric Antigen Receptors are Active AgainstBCMA Expressing Cells

T-cells from normal donors were transduced with the different APRIL CARsand tested against SupT1 cells either wild-type, or engineered toexpress BCMA and TACI. Several different assays were used to determinefunction. A classical chromium release assay was performed. Here, thetarget cells (the SupT1 cells) were labelled with 51Cr and mixed witheffectors (the transduced T-cells) at different ratio. Lysis of targetcells was determined by counting 51Cr in the co-culture supernatant(FIG. 28A shows the cumulative data).

In addition, supernatant from T-cells cultured 1:1 with SupT1 cells wasassayed by ELISA for Interferon-gamma (FIG. 28B shows cumulative data).Measurement of T-cell expansion after one week of co-culture with SupT1cells was also performed (FIG. 28C). T-cells were counted byflow-cytometry calibrated with counting beads. These experimental datashow that APRIL based CARs can kill BCMA expressing targets. Further,these data show that CARs based on the CD8 stalk or IgG1 hinge performedbetter than the Fc-pvaa based CAR.

Example 14 Functional Analysis of the AND Pate in Primary Cells

PBMCs were isolated from blood and stimulated using PHA and IL-2. Twodays later the cells were transduced on retronectin coated plates withretro virus containing the CD19:CD33 AND gate construct. On day 5 theexpression level of the two CARs translated by the AND gate constructwas evaluated via flow cytometry and the cells were depleted of CD56+cells (predominantly NK cells). On day 6 the PBMCs were placed in aco-culture with target cells at a 1:2 effector to target cell ratio. Onday 8 the supernatant was collected and analysed for IFN-gamma secretionvia ELISA (FIG. 29).

These data demonstrate that the AND gate functions in primary cells.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, cell biology or related fields are intended to bewithin the scope of the following claims.

1. A T cell or natural killer (NK) cell which co-expresses a firstchimeric antigen receptor (CAR) and second CAR at the cell surface, eachCAR comprising: (i) an antigen-binding domain; (ii) a spacer (iii) atrans-membrane domain; and (iv) an endodomain wherein the antigenbinding domains of the first and second CARs bind to different antigens,wherein the spacer of the first CAR is different to the spacer of thesecond CAR and wherein one of the first or second CARs is an activatingCAR comprising an activating endodomain and the other CAR is aninhibitory CAR comprising a ligation-off inhibitory endodomain.
 2. The Tor NK cell according to claim 1, wherein the spacer of the first CAR hasa different length and/or charge and/or size and/or configuration and/orglycosylation of the spacer of the second CAR, such that when the firstCAR and the second CAR bind their respective target antigens, the firstCAR and second CAR become spatially separated on the T cell membrane. 3.The T or NK cell according to claim 2, wherein either the first spaceror the second spacer comprises a CD8 stalk and the other spacercomprises the hinge, CH2 and CH3 domain of IgG1.
 4. The T or NK cellaccording to claim 2, wherein one of the first or second CARs is anactivating CAR comprising an activating endodomain, and the other CAR isan inhibitory CAR comprising a ligation-off inhibitory endodomain, whichinhibitory CAR inhibits T-cell activation by the activating CAR in theabsence of inhibitory CAR ligation, but does not significantly inhibitT-cell activation by the activating CAR when the inhibitory CAR isligated.
 5. The T or NK cell according to claim 4, wherein theinhibitory endodomain comprises all or part of the endodomain from CD148or CD45.
 6. The T or NK cell according to claim 4, wherein theantigen-binding domain of the first CAR binds CD5 and theantigen-binding domain of the second CAR binds CD19.
 7. (canceled)
 8. Anucleic acid sequence encoding both first and second chimeric antigenreceptors (CARs) as defined in claim
 1. 9. The nucleic acid sequenceaccording to claim 8, which has the following structure:AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 in which AgB1 is anucleic acid sequence encoding the antigen-binding domain of the firstCAR; spacer 1 is a nucleic acid sequence encoding the spacer of thefirst CAR; TM1 is a nucleic acid sequence encoding the transmembranedomain of the first CAR; endo 1 is a nucleic acid sequence encoding theendodomain of the first CAR; coexpr is a nucleic acid sequence enablingco-expression of both CARs AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CAR; whichnucleic acid sequence, when expressed in a T cell, encodes a polypeptidewhich is cleaved at the cleavage site such that the first and secondCARs are co-expressed at the T cell surface.
 10. The nucleic acidsequence according to claim 9, wherein coexpr encodes a sequencecomprising a self-cleaving peptide.
 11. The nucleic acid sequenceaccording to claim 9, wherein alternative codons are used in regions ofsequence encoding the same or similar amino acid sequences, in order toavoid homologous recombination.
 12. A kit which comprises (i) a firstnucleic acid sequence encoding the first chimeric antigen receptor (CAR)as defined in claim 1, which nucleic acid sequence has the followingstructure: AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acidsequence encoding the antigen-binding domain of the first CAR; spacer 1is a nucleic acid sequence encoding the spacer of the first CAR; TM1 isa nucleic acid sequence encoding the transmembrane domain of the firstCAR; endo 1 is a nucleic acid sequence encoding the endodomain of thefirst CAR; and (ii) a second nucleic acid sequence encoding the secondchimeric antigen receptor (CAR) as defined in claim 1, which nucleicacid sequence has the following structure: AgB2-spacer2-TM2-endo2 AgB2is a nucleic acid sequence encoding the antigen-binding domain of thesecond CAR; spacer 2 is a nucleic acid sequence encoding the spacer ofthe second CAR; TM2 is a nucleic acid sequence encoding thetransmembrane domain of the second CAR; endo 2 is a nucleic acidsequence encoding the endodomain of the second CAR.
 13. The kitcomprising: a first vector which comprises the first nucleic acidsequence as defined in claim 12; and a second vector which comprises thefirst nucleic acid sequence as defined in claim
 12. 14. The kitaccording to claim 13, wherein the vectors are integrating viral vectorsor transposons.
 15. A vector comprising a nucleic acid sequenceaccording to claim
 8. 16. The vector according to claim 15 which is aretroviral vector or a lentiviral vector or a transposon.
 17. A methodfor making a T or NK cell according to claim 1, which comprises the stepof introducing: a nucleic acid sequence according to claim 8; a firstnucleic acid sequence and a second nucleic acid sequence as defined inclaim 12; and/or a first vector and a second vector as defined in claim13 or a vector according to claim 15, into a T or NK cell.
 18. Themethod according to claim 17, wherein the T or NK cell is from a sampleisolated from a subject.
 19. A pharmaceutical composition comprising aplurality of T or NK cells according to claim
 1. 20. A method fortreating and/or preventing a disease, which comprises the step ofadministering a pharmaceutical composition according to claim 19 to asubject.
 21. The method according to claim 20, which comprises thefollowing steps: (i) isolation of a T cell-containing sample from asubject; (ii) transduction or transfection of the T cells with: anucleic acid sequence according to claim 8; a first nucleic acidsequence and a second nucleic acid sequence as defined in claim 12; afirst vector and a second vector as defined in claim 13 or a vectoraccording to claim 15; and (iii) administering the T cells from (ii) toa the subject.
 22. The method according to claim 20, wherein the diseaseis a cancer. 23-26. (canceled)